scholarly journals Mass allelic exchange: enabling sexual genetics in Escherichia coli

2020 ◽  
Author(s):  
Varnica Khetrapal ◽  
Liyana Ow Yong ◽  
Swaine L. Chen
Keyword(s):  
Linkage Analysis ◽  
Allelic Variation ◽  
Phenotypic Diversity ◽  
F1 Hybrids ◽  
Natural Phenomenon ◽  
Loss Of Function ◽  
E Coli ◽  
Allelic Exchange ◽  
Strong Negative Selection ◽  
Single Operon

AbstractDespite dramatic advances in genomics, connecting genotypes to phenotypes is still challenging. Sexual genetics combined with linkage analysis is a powerful solution to this problem but generally unavailable in bacteria. We build upon a strong negative selection system to invent Mass Allelic Exchange (MAE), which enables hybridization of arbitrary (including pathogenic) strains of E. coli. MAE reimplements the natural phenomenon of random crossovers, enabling classical linkage analysis. We demonstrate the utility of MAE with virulence-related gain-of-function screens, discovering that transfer of a single operon from a uropathogenic strain is sufficient for enabling a commensal E. coli to form large intracellular bacterial collections within bladder epithelial cells. MAE thus enables assaying natural allelic variation in E. coli (and potentially other bacteria), complementing existing loss-of-function genomic techniques.One Sentence SummaryWe create F1 hybrids of E. coli using MAE, bringing the power of linkage analysis to bear on phenotypic diversity (including virulence)

scholarly journals Production, characterization, and cross-reactivity of a polyclonal antibody against Arabidopsis TARGET OF RAPAMYCIN

2019 ◽  
Vol 62 (1) ◽  
Author(s):  
Gyeong-Im Shin ◽  
Sun Young Moon ◽  
Song Yi Jeong ◽  
Myung Geun Ji ◽  
Joon-Yung Cha ◽  
...  
Keyword(s):  
Molecular Mechanisms ◽  
Cross Reactivity ◽  
Target Of Rapamycin ◽  
Loss Of Function ◽  
Anticancer Activities ◽  
Purification Method ◽  
E Coli ◽  
Related Family ◽  
Large Gene ◽  
Truncated Variants

AbstractTARGET OF RAPAMYCIN (TOR), a member of the phosphatidylinositol 3-kinase-related family of protein kinases, is encoded by a single, large gene and is evolutionarily conserved in all eukaryotes. TOR plays a role as a master regulator that integrates nutrient, energy, and stress signaling to orchestrate development. TOR was first identified in yeast mutant screens, as its mutants conferred resistance to rapamycin, an antibiotic with immunosuppressive and anticancer activities. In Arabidopsis thaliana, the loss-of-function tor mutant displays embryo lethality, but the precise mechanisms of TOR function are still unknown. Moreover, a lack of reliable molecular and biochemical assay tools limits our ability to explore TOR functions in plants. Here, we produced a polyclonal α-TOR antibody using two truncated variants of TOR (1–200 and 1113–1304 amino acids) as antigens because recombinant full-length TOR is challenging to express in Escherichia coli. Recombinant His-TOR1−200 and His-TOR1113−1304 proteins were individually expressed in E. coli, and a mixture of proteins (at a 1:1 ratio) was used for immunizing rabbits. Antiserum was purified by an antigen-specific purification method, and the purified polyclonal α-TOR antibody successfully detected endogenous TOR proteins in wild-type Arabidopsis and TOR orthologous in major crop plants, including tomato, maize, and alfalfa. Moreover, our α-TOR antibody is useful for coimmunoprecipitation assays. In summary, we generated a polyclonal α-TOR antibody that detects endogenous TOR in various plant species. Our antibody could be used in future studies to determine the precise molecular mechanisms of TOR, which has largely unknown multifunctional roles in plants.

scholarly journals The haplolethality paradox of the wupA gene in Drosophila

PLoS Genetics ◽  
2021 ◽  
Vol 17 (3) ◽  
pp. e1009108
Author(s):  
Sergio Casas-Tintó ◽  
Alberto Ferrús
Keyword(s):  
Cell Proliferation ◽  
Troponin I ◽  
Gene Expression Regulation ◽  
Nuclear Translocation ◽  
Normal Function ◽  
Protein Isoforms ◽  
Large Set ◽  
Loss Of Function ◽  
Induced Mutations ◽  
Strong Negative Selection

Haplolethals (HL) are regions of diploid genomes that in one dose are fatal for the organism. Their biological meaning is obscure because heterozygous loss-of-function mutations result in dominant lethality (DL) and, consequently, should be under strong negative selection. We report an in depth study of the HL associated to the gene wings up A (wupA). It encodes 13 transcripts (A-M) that yield 11 protein isoforms (A-K) of Troponin I (TnI). They are functionally diverse in their control of muscle contraction, cell polarity and cell proliferation. Isoform K transfers to the nucleus where it increases transcription of the cell proliferation related genes CDK2, CDK4, Rap and Rab5. The nuclear translocation of isoform K is prevented by the co-expression of A or B isoforms, which illustrates isoform interactions. The corresponding DL mutations are, either DNA rearrangements clustered towards the gene 3’ end, thus affecting the genomic organization of all transcripts, or CRISPR-induced mutations in one of the two ATG sites which eliminate a subset of wupA products. The joint elimination of isoforms C, F, G and H, however, do not cause DL phenotypes. Genetically driven expression of single isoforms rescue neither DL nor any of the mutants known in the gene, suggesting that normal function requires properly regulated expression of specific combinations, rather than single, TnI isoforms. We conclude that the wupA associated HL results from the combined haploinsufficiency of a large set of TnI isoforms. The qualitative and quantitative normal expression of which, requires the chromosomal integrity of the wupA genomic region. Since all fly TnI isoforms are encoded in the same gene, its HL condition becomes unavoidable. These wupA features are comparable to those of dpp, the only other HL studied to some extent, and reveal a scenario of strict dosage dependence with implications for gene expression regulation and splitting.

scholarly journals Genomewide Mutational Diversity in Escherichia coli Population Evolving in Prolonged Stationary Phase

mSphere ◽  
2017 ◽  
Vol 2 (3) ◽  
Author(s):  
Savita Chib ◽  
Farhan Ali ◽  
Aswin Sai Narain Seshasayee
Keyword(s):  
Escherichia Coli ◽  
Genetic Diversity ◽  
Stationary Phase ◽  
Phenotypic Diversity ◽  
Model System ◽  
Content Type ◽  
Neutral Drift ◽  
E Coli ◽  
Evolution By Natural Selection ◽  
Over Time

ABSTRACT Prolonged stationary phase in bacteria, contrary to its name, is highly dynamic, with extreme nutrient limitation as a predominant stress. Stationary-phase cultures adapt by rapidly selecting a mutation(s) that confers a growth advantage in stationary phase (GASP). The phenotypic diversity of starving E. coli populations has been studied in detail; however, only a few mutations that accumulate in prolonged stationary phase have been described. This study documented the spectrum of mutations appearing in Escherichia coli during 28 days of prolonged starvation. The genetic diversity of the population increases over time in stationary phase to an extent that cannot be explained by random, neutral drift. This suggests that prolonged stationary phase offers a great model system to study adaptive evolution by natural selection. Prolonged stationary phase is an approximation of natural environments presenting a range of stresses. Survival in prolonged stationary phase requires alternative metabolic pathways for survival. This study describes the repertoire of mutations accumulating in starving Escherichia coli populations in lysogeny broth. A wide range of mutations accumulates over the course of 1 month in stationary phase. Single nucleotide polymorphisms (SNPs) constitute 64% of all mutations. A majority of these mutations are nonsynonymous and are located at conserved loci. There is an increase in genetic diversity in the evolving populations over time. Computer simulations of evolution in stationary phase suggest that the maximum frequency of mutations observed in our experimental populations cannot be explained by neutral drift. Moreover, there is frequent genetic parallelism across populations, suggesting that these mutations are under positive selection. Finally, functional analysis of mutations suggests that regulatory mutations are frequent targets of selection. IMPORTANCE Prolonged stationary phase in bacteria, contrary to its name, is highly dynamic, with extreme nutrient limitation as a predominant stress. Stationary-phase cultures adapt by rapidly selecting a mutation(s) that confers a growth advantage in stationary phase (GASP). The phenotypic diversity of starving E. coli populations has been studied in detail; however, only a few mutations that accumulate in prolonged stationary phase have been described. This study documented the spectrum of mutations appearing in Escherichia coli during 28 days of prolonged starvation. The genetic diversity of the population increases over time in stationary phase to an extent that cannot be explained by random, neutral drift. This suggests that prolonged stationary phase offers a great model system to study adaptive evolution by natural selection.

scholarly journals Identification of a Streptococcus pneumoniae Gene Locus Encoding Proteins of an ABC Phosphate Transporter and a Two-Component Regulatory System

1999 ◽  
Vol 181 (4) ◽  
pp. 1126-1133 ◽  
Author(s):  
Rodger Novak ◽  
Anje Cauwels ◽  
Emmanuelle Charpentier ◽  
Elaine Tuomanen
Keyword(s):  
Response Regulator ◽  
Regulatory System ◽  
Phosphate Limitation ◽  
Pho Regulon ◽  
Loss Of Function ◽  
Gene Encoding ◽  
E Coli ◽  
Two Component ◽  
Pst System

ABSTRACT The Escherichia coli Pst system belongs to the family of ABC transporters. It is part of a phosphate (PHO) regulon which is regulated by extracellular phosphate. Under conditions of phosphate limitation, the response regulator PhoB is phosphorylated by the histidine kinase PhoR and binds to promoters that share a consensus PHO box. Under conditions of phosphate excess, PhoR, Pst, and PhoU downregulate the PHO regulon. Screening of a library of pneumococcal mutants with defects in exported proteins revealed a putative two-component regulatory system, PnpR-PnpS, and a downstream ABC transporter, similar to the Pst system in E. coli including a gene encoding a PhoU protein. Similar to E. coli, mutagenesis of the ATP-binding cassette gene, pstB, resulted in decreased uptake of phosphate. The effects of the loss of the pneumococcal Pst system extended to decreased transformation and lysis. Withdrawal of phosphate led to transformation deficiency in the parent strain R6x but not to penicillin tolerance, suggesting that reduced bacterial death was independent of phosphate. None of these phenotypes was observed in the pneumococcal loss-of-function mutantphoU. By using a lacZ reporter construct, it was demonstrated that expression of the two-component regulatory system PnpR-PnpS was not influenced by different concentrations of phosphate. These results suggest a more complex role of the Pst system in pneumococcal physiology than in that of E. coli.

scholarly journals Human P2X7 Pore Function Predicts Allele Linkage Disequilibrium

2006 ◽  
Vol 52 (6) ◽  
pp. 995-1004 ◽  
Author(s):  
Loren C Denlinger ◽  
Douglas B Coursin ◽  
Kathleen Schell ◽  
Giuditta Angelini ◽  
Dawn N Green ◽  
...  
Keyword(s):  
Linkage Disequilibrium ◽  
Linkage Analysis ◽  
Whole Blood ◽  
Interleukin 10 ◽  
Genomic Variation ◽  
Loss Of Function ◽  
Roc Curve Analysis ◽  
Activity Data ◽  
Diagnostic Threshold ◽  
Sequence Variations

Abstract Background: Innate immune response amplification is achieved by leukocyte expression of the purinergic nucleotide receptor P2X7, an extracellular nucleotide-gated pore. Previously, low P2X7 pore activity in whole blood was associated with loss-of-function genotypes in correlation with a decreased ratio of lipopolysaccharide-stimulated tumor necrosis factor-α to interleukin-10, of relevance to a variety of infectious and inflammatory disorders. We hypothesized that evaluation of participants with discordance between the P2X7 genotype and pore status would disclose additional alleles, linkage disequilibrium, and novel functional correlates of genotype to phenotype. Methods: Comparison of whole-blood pore results with restriction fragment length polymorphism data for known loss-of-function genotypes from 200 healthy participants optimized the diagnostic threshold for low pore activity by ROC curve analysis. We identified novel alleles and inferred haplotypes by sequencing outlier genomic templates and by linkage analysis. Results: With a refined threshold of low activity, a normal pore result had only a 2% probability of association with known loss-of-function variants. By contrast, the positive predictive value of low pore activity was 59% for identifying known alleles. DNA samples from this discordant group contained 28 P2X7 sequence variations. Linkage analysis demonstrated that A1513C, T1729A, and G946A are inherited independently from one another, although these loss-of-function variants are in disequilibrium with other alleles. When we segregated pore activity data according to genotypes, nonsynonymous sequence variations (G474A and A1405G) appeared to exhibit modulatory effects on P2X7 pore activity. Conclusions: Direct analysis of pore activity demonstrates functional interactions between P2X7 alleles. The performance characteristics of the whole-blood pore assay enables correlation of genomic variation with concomitant investigation of functional performance in clinical studies.

scholarly journals Acetate and glycerol are not uniquely suited for the evolution of cross-feeding in E. coli

2020 ◽  
Vol 16 (11) ◽  
pp. e1008433
Author(s):  
Magdalena San Roman ◽  
Andreas Wagner
Keyword(s):  
Carbon Source ◽  
Experimental Evolution ◽  
Phenotypic Diversity ◽  
Carbon Sources ◽  
Metabolic Modeling ◽  
Metabolic Changes ◽  
E Coli ◽  
Biochemical Pathways ◽  
Genome Scale ◽  
Feeding Interactions

The evolution of cross-feeding among individuals of the same species can help generate genetic and phenotypic diversity even in completely homogeneous environments. Cross-feeding Escherichia coli strains, where one strain feeds on a carbon source excreted by another strain, rapidly emerge during experimental evolution in a chemically minimal environment containing glucose as the sole carbon source. Genome-scale metabolic modeling predicts that cross-feeding of 58 carbon sources can emerge in the same environment, but only cross-feeding of acetate and glycerol has been experimentally observed. Here we use metabolic modeling to ask whether acetate and glycerol cross-feeding are especially likely to evolve, perhaps because they require less metabolic change, and thus perhaps also less genetic change than other cross-feeding interactions. However, this is not the case. The minimally required metabolic changes required for acetate and glycerol cross feeding affect dozens of chemical reactions, multiple biochemical pathways, as well as multiple operons or regulons. The complexity of these changes is consistent with experimental observations, where cross-feeding strains harbor multiple mutations. The required metabolic changes are also no less complex than those observed for multiple other of the 56 cross feeding interactions we study. We discuss possible reasons why only two cross-feeding interactions have been discovered during experimental evolution and argue that multiple new cross-feeding interactions may await discovery.

scholarly journals Identification and Allelic Variation of Genes Involved in the Potato Glycoalkaloid Biosynthetic Pathway

2012 ◽  
Author(s):  
Idit Ginzberg ◽  
Richard E. Veilleux ◽  
James G. Tokuhisa
Keyword(s):  
Transgenic Plants ◽  
Candidate Genes ◽  
Biosynthetic Pathway ◽  
Developmental Stages ◽  
Allelic Variation ◽  
Mevalonic Acid ◽  
Transcript Abundance ◽  
Primary Metabolism ◽  
E Coli ◽  
Isoprenoid Pathway

Steroidal glycoalkaloids (SGAs) are secondary metabolites being part of the plant defense response. The two major SGAs in cultivated potato (Solanum tuberosum) are α-chaconine and α-solanine, which exhibit strong cellular lytic properties and inhibit acetylcholinesterase activity, and are poisonous at high concentrations for humans. As SGAs are not destroyed during cooking and frying commercial cultivars have been bred to contain low levels, and their content in tubers should not exceed 20 mg/100 g fresh weight. However, environmental factors can increase tuber SGA content above the safe level. The focus of the proposed research was to apply genomic approaches to identify candidate genes that control potato SGA content in order to develop tools for potato improvement by marker-assisted selection and/or transgenic approaches. To this end, the objectives of the proposal included identification of genes, metabolic intermediates and allelic variations in the potato SGAbiosynthetic pathway.   The SGAs are biosynthesized by the sterol branch of the mevalonic acid/isoprenoid pathway. Transgenic potato plants that overexpress 3-hydroxy-3-methylglutaryl-CoA reductase 1 (HMG1) or squalene synthase 1 (SQS1), key enzymes of the mevalonic acid/isoprenoid pathway, exhibited elevated levels of solanine and chaconine as well as induced expression of genes downstream the pathway. These results suggest of coordinated regulation of isoprenoid (primary) metabolism and SGA secondary metabolism. The transgenic plants were further used to identify new SGA-related candidate genes by cDNA-AFLP approach and a novel glycosyltransferase was isolated. In addition, genes involved in phytosterol biosynthesis may have dual role and synthesize defense-related steroidal metabolites, such as SGAs, via lanosterol pathway. Potato lanosterol synthase sequence (LAS) was isolated and used to prepare transgenic plants with overexpressing and silencing constructs. Plants are currently being analyzed for SGA content.   The dynamics of SGA accumulation in the various organs of a potato species with high SGA content gave insights into the general regulation of SGA abundance. Leaf SGA levels in S. chacoense were 10 to 20-fold greater than those of S. tuberosum. The leptines, SGAs with strong antifeedant properties against Colorado potato beetles, were present in all aerial tissues except for early and mid-developmental stages of above ground stolons, and accounted for the high SGA content of S. chacoense. These results indicate the presence of regulatory mechanisms in most tissues except in stolons that limit the levels of α-solanine and α-chaconine and confine leptine accumulation to the aerial tissues.   The genomes of cultivated and wild potato contain a 4-member gene family coding for SQS. Three orthologs were cloned as cDNAs from S. chacoense and heterologously expressed in E. coli. Squalene accumulated in all E. coli lines transformed with each of the three gene constructs. Differential transcript abundance in various organs and amino acid sequence differences in the conserved domains of three isoenzymes indicate subfunctionalization of SQS activity and triterpene/sterol metabolism.   Because S. chacoense and S. phureja differ so greatly for presence and accumulation of SGAs, we selected four candidate genes from different points along the biosynthetic pathway to determine if chcor phuspecific alleles were associated with SGA expression in a segregating interspecific diploid population. For two of the four genes (HMG2 and SGT2) F2 plants with chcalleles expressed significantly greater total SGAs compared with heterozygotes and those with phualleles. Although there are other determinants of SGA biosynthesis and composition in potato, the ability of allelic states at two genes to affect SGA levels confirms some of the above transgenic work where chcalleles at two other loci altered SGA expression in Desiree.   Present results reveal new opportunities to manipulate triterpene/sterol biosynthesis in more targeted ways with the objective of altering SGA content for both human health concerns and natural pesticide content without disrupting the essential metabolism and function of the phytosterol component of the membranes and the growth regulating brassinosteroids.

scholarly journals Heat-induced transposition leads to rapid drought resistance of Arabidopsis thaliana

2021 ◽  
Author(s):  
Michael Thieme ◽  
Arthur Brêchet ◽  
Yann Bourgeois ◽  
Bettina Keller ◽  
Etienne Bucher ◽  
...  
Keyword(s):  
Arabidopsis Thaliana ◽  
Drought Resistance ◽  
Phenotypic Diversity ◽  
Plant Evolution ◽  
Loss Of Function ◽  
Copy Numbers ◽  
Drastic Increase ◽  
In The Wild ◽  
Rate Of Photosynthesis

Plant genomes comprise a vast diversity of transposable elements (TEs) (Tenaillon et al. 2010)⁠. While their uncontrolled proliferation can have fatal consequences for their host, there is strong evidence for their importance in fueling genetic diversity and plant evolution (Baduel et al. 2021)⁠. However, the number of studies addressing the role of TEs in this process is limited. Here we show that the heat-induced burst of a low-copy TE increases phenotypic diversity and leads to the rapid emergence of more drought-resistant individuals of Arabidopsis thaliana. We exposed TE-high-copy-(hc)lines (Thieme et al. 2017)⁠ with up to ~8 fold increased copy numbers of the heat-responsive ONSEN-TE (AtCOPIA78) (Ito et al. 2011; Cavrak et al. 2014; Tittel-Elmer et al. 2010)⁠ in the wild type background to desiccation as a straightforward and highly relevant selection pressure. We found evidence for a drastic increase of drought resistance in five out of the 23 tested hc-lines and further pinpoint one of the causative mutations to an exonic ONSEN-insertion in the ribose-5-phosphate-isomerase 2 gene. This loss-of-function mutation resulted in a decreased rate of photosynthesis and water consumption. This is one of the rare examples (Esnault et al. 2019)⁠ experimentally demonstrating the adaptive potential of mobilized stress-responsive TEs in eukaryotes. Our results further shed light on the complex relationship between mobile elements and their hosts and substantiate the importance of TE-mediated loss-of-function mutations in stress adaptation, particularly with respect to global warming.

scholarly journals Identification of disease-linked hyperactivating mutations in UBE3A through large-scale functional variant analysis

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Kellan P. Weston ◽  
Xiaoyi Gao ◽  
Jinghan Zhao ◽  
Kwang-Soo Kim ◽  
Susan E. Maloney ◽  
...  
Keyword(s):  
Angelman Syndrome ◽  
Large Scale ◽  
Phenotypic Diversity ◽  
Function Analysis ◽  
Genetic Disorders ◽  
Neurodevelopmental Disorder ◽  
Loss Of Function ◽  
Monogenic Disorders ◽  
Functional Variant ◽  
Variant Analysis

AbstractThe mechanisms that underlie the extensive phenotypic diversity in genetic disorders are poorly understood. Here, we develop a large-scale assay to characterize the functional valence (gain or loss-of-function) of missense variants identified in UBE3A, the gene whose loss-of-function causes the neurodevelopmental disorder Angelman syndrome. We identify numerous gain-of-function variants including a hyperactivating Q588E mutation that strikingly increases UBE3A activity above wild-type UBE3A levels. Mice carrying the Q588E mutation exhibit aberrant early-life motor and communication deficits, and individuals possessing hyperactivating UBE3A variants exhibit affected phenotypes that are distinguishable from Angelman syndrome. Additional structure-function analysis reveals that Q588 forms a regulatory site in UBE3A that is conserved among HECT domain ubiquitin ligases and perturbed in various neurodevelopmental disorders. Together, our study indicates that excessive UBE3A activity increases the risk for neurodevelopmental pathology and suggests that functional variant analysis can help delineate mechanistic subtypes in monogenic disorders.

scholarly journals Harnessing population-specific protein truncating variants to improve the annotation of loss-of-function alleles

2020 ◽  
Author(s):  
Rostislav K. Skitchenko ◽  
Julia S. Kornienko ◽  
Evgeniia M. Maksiutenko ◽  
Andrey S. Glotov ◽  
Alexander V. Predeus ◽  
...  
Keyword(s):  
Population Genomics ◽  
False Positive Rate ◽  
Threshold Value ◽  
Specific Protein ◽  
Human Populations ◽  
Loss Of Function ◽  
Human Genes ◽  
Strong Negative Selection ◽  
Disease Penetrance ◽  
Positive Rate

AbstractAccurate annotation of putative loss-of-function (pLoF) variants is an important problem in human genomics and disease, which recently drew substantial attention. Since such variants in disease-related genes are under strong negative selection, their frequency across major ancestral groups is expected to be highly similar. In this study, we tested this assumption by systematically assessing the presence of highly population-specific protein-truncating variants (PTVs) in human genes using latest population-scale data. We discovered an unexpectedly high incidence of population-specific PTVs in all major ancestral groups. This does not conform to a recently proposed model, indicating either systemic differences in disease penetrance in different human populations, or a failure of current annotation criteria to accurately predict the loss-of-function potential of PTVs. We show that low-confidence pLoF variants are enriched in genes with non-uniform PTV count distribution, and developed a computational tool called LoFfeR that can efficiently predict tolerated pLoF variants. To evaluate the performance of LoFfeR, we use a set of known pathogenic and benign PTVs from the ClinVar database, and show that LoFfeR allows for a more accurate annotation of low-confidence pLoF variants compared to existing methods. Notably, only 4.4% of protein-truncating gnomAD SNPs in canonical transcripts can be filtered out using a recommended threshold value of the recently proposed pext score, while up to 10.9% of such variants are filtered using LoFfeR with the same false positive rate. Hence, we believe that LoFfeR can be used for additional filtering of low-confidence pLoF variants in population genomics and medical genetics studies.

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