scholarly journals Collateral Toxicity Limits the Evolution of Bacterial Release Factor 2 toward Total Omnipotence

2020 ◽  
Vol 37 (10) ◽  
pp. 2918-2930
Author(s):  
Hind Abdalaal ◽  
Shreya Pundir ◽  
Xueliang Ge ◽  
Suparna Sanyal ◽  
Joakim Näsvall
Keyword(s):  
Copy Number ◽  
Gene Evolution ◽  
Gene Copy Number ◽  
In Vitro Translation ◽  
Gene Copy ◽  
Release Factor ◽  
Bacterial Growth Rate ◽  
New Genes ◽  
Peptide Release

Abstract When new genes evolve through modification of existing genes, there are often tradeoffs between the new and original functions, making gene duplication and amplification necessary to buffer deleterious effects on the original function. We have used experimental evolution of a bacterial strain lacking peptide release factor 1 (RF1) in order to study how peptide release factor 2 (RF2) evolves to compensate the loss of RF1. As expected, amplification of the RF2-encoding gene prfB to high copy number was a rapid initial response, followed by the appearance of mutations in RF2 and other components of the translation machinery. Characterization of the evolved RF2 variants by their effects on bacterial growth rate, reporter gene expression, and in vitro translation termination reveals a complex picture of reduced discrimination between the cognate and near-cognate stop codons and highlights a functional tradeoff that we term “collateral toxicity.” We suggest that this type of tradeoff may be a more serious obstacle in new gene evolution than the more commonly discussed evolutionary tradeoffs between “old” and “new” functions of a gene, as it cannot be overcome by gene copy number changes. Further, we suggest a model for how RF2 autoregulation responds to alterations in the demand not only for RF2 activity but also for RF1 activity.

scholarly journals Leishmania naiffi and Leishmania guyanensis reference genomes highlight genome structure and gene evolution in the Viannia subgenus

2018 ◽  
Vol 5 (4) ◽  
pp. 172212 ◽  
Author(s):  
Simone Coughlan ◽  
Ali Shirley Taylor ◽  
Eoghan Feane ◽  
Mandy Sanders ◽  
Gabriele Schonian ◽  
...  
Keyword(s):  
Copy Number ◽  
Structural Changes ◽  
Gene Evolution ◽  
Genome Structure ◽  
Gene Copy Number ◽  
Gene Copy ◽  
Individual Species ◽  
Short Sequence ◽  
Origin And Evolution ◽  
Reference Genomes

The unicellular protozoan parasite Leishmania causes the neglected tropical disease leishmaniasis, affecting 12 million people in 98 countries. In South America, where the Viannia subgenus predominates, so far only L. ( Viannia ) braziliensis and L. ( V. ) panamensis have been sequenced, assembled and annotated as reference genomes. Addressing this deficit in molecular information can inform species typing, epidemiological monitoring and clinical treatment. Here, L. ( V. ) naiffi and L. ( V. ) guyanensis genomic DNA was sequenced to assemble these two genomes as draft references from short sequence reads. The methods used were tested using short sequence reads for L. braziliensis M2904 against its published reference as a comparison. This assembly and annotation pipeline identified 70 additional genes not annotated on the original M2904 reference. Phylogenetic and evolutionary comparisons of L. guyanensis and L. naiffi with 10 other Viannia genomes revealed four traits common to all Viannia : aneuploidy, 22 orthologous groups of genes absent in other Leishmania subgenera, elevated TATE transposon copies and a high NADH-dependent fumarate reductase gene copy number. Within the Viannia , there were limited structural changes in genome architecture specific to individual species: a 45 Kb amplification on chromosome 34 was present in all bar L. lainsoni , L. naiffi had a higher copy number of the virulence factor leishmanolysin, and laboratory isolate L. shawi M8408 had a possible minichromosome derived from the 3’ end of chromosome 34 . This combination of genome assembly, phylogenetics and comparative analysis across an extended panel of diverse Viannia has uncovered new insights into the origin and evolution of this subgenus and can help improve diagnostics for leishmaniasis surveillance.

scholarly journals Leishmania Genome Dynamics during Environmental Adaptation Reveal Strain-Specific Differences in Gene Copy Number Variation, Karyotype Instability, and Telomeric Amplification

mBio ◽  
2018 ◽  
Vol 9 (6) ◽  
Author(s):  
Giovanni Bussotti ◽  
Evi Gouzelou ◽  
Mariana Côrtes Boité ◽  
Ihcen Kherachi ◽  
Zoubir Harrat ◽  
...  
Keyword(s):  
Environmental Change ◽  
Copy Number ◽  
Gene Copy Number ◽  
Environmental Adaptation ◽  
Gene Copy ◽  
Protozoan Parasites ◽  
Content Type ◽  
Intrinsic Factors ◽  
Genomic Adaptation

ABSTRACT Protozoan parasites of the genus Leishmania adapt to environmental change through chromosome and gene copy number variations. Only little is known about external or intrinsic factors that govern Leishmania genomic adaptation. Here, by conducting longitudinal genome analyses of 10 new Leishmania clinical isolates, we uncovered important differences in gene copy number among genetically highly related strains and revealed gain and loss of gene copies as potential drivers of long-term environmental adaptation in the field. In contrast, chromosome rather than gene amplification was associated with short-term environmental adaptation to in vitro culture. Karyotypic solutions were highly reproducible but unique for a given strain, suggesting that chromosome amplification is under positive selection and dependent on species- and strain-specific intrinsic factors. We revealed a progressive increase in read depth towards the chromosome ends for various Leishmania isolates, which may represent a nonclassical mechanism of telomere maintenance that can preserve integrity of chromosome ends during selection for fast in vitro growth. Together our data draw a complex picture of Leishmania genomic adaptation in the field and in culture, which is driven by a combination of intrinsic genetic factors that generate strain-specific phenotypic variations, which are under environmental selection and allow for fitness gain. IMPORTANCE Protozoan parasites of the genus Leishmania cause severe human and veterinary diseases worldwide, termed leishmaniases. A hallmark of Leishmania biology is its capacity to adapt to a variety of unpredictable fluctuations inside its human host, notably pharmacological interventions, thus, causing drug resistance. Here we investigated mechanisms of environmental adaptation using a comparative genomics approach by sequencing 10 new clinical isolates of the L. donovani, L. major, and L. tropica complexes that were sampled across eight distinct geographical regions. Our data provide new evidence that parasites adapt to environmental change in the field and in culture through a combination of chromosome and gene amplification that likely causes phenotypic variation and drives parasite fitness gains in response to environmental constraints. This novel form of gene expression regulation through genomic change compensates for the absence of classical transcriptional control in these early-branching eukaryotes and opens new venues for biomarker discovery.

scholarly journals Resistance to Antimalarials in Southeast Asia and Genetic Polymorphisms in pfmdr1

2003 ◽  
Vol 47 (8) ◽  
pp. 2418-2423 ◽  
Author(s):  
Amy L. Pickard ◽  
Chansuda Wongsrichanalai ◽  
Anne Purfield ◽  
Deborah Kamwendo ◽  
Kathryn Emery ◽  
...  
Keyword(s):  
Southeast Asia ◽  
Copy Number ◽  
Gene Copy Number ◽  
Public Health Problem ◽  
Prevalence Ratio ◽  
Chloroquine Resistance ◽  
Gene Copy ◽  
Drug Resistant ◽  
Wild Type

ABSTRACT Resistance to antimalarial drugs is a public health problem worldwide. Molecular markers for drug-resistant malaria, such as pfcrt and pfmdr1 polymorphisms, could serve as useful surveillance tools. To evaluate this possibility, sequence polymorphisms in pfcrt (position 76) and pfmdr1 (positions 86, 184, 1034, 1042, and 1246) and in vitro drug sensitivities were measured for 65 Plasmodium falciparum isolates from Thailand, Myanmar, Vietnam, and Bangladesh. The pfcrt Thr76 polymorphism was present in 97% of samples, consistent with observations that chloroquine resistance is well established in this region. Polymorphisms in pfmdr1 clustered into four specific patterns: the wild type (category I), a Tyr86 polymorphism only (category II), a Phe184 polymorphism only (category III), and Phe184 in combination with Cys1034 and/or Asp1042 (category IV). Isolates in categories I and III were more sensitive to chloroquine and more resistant to mefloquine, artesunate, and artemisinin than isolates in categories II and IV (P ≤ 0.01). Mefloquine resistance was significantly more common in category I and III isolates than in category II and IV isolates, with a prevalence ratio of 14.95 (95% confidence interval, 3.88 to 57.56). These categories identified mefloquine resistance with a sensitivity and a specificity of 94 and 91%, respectively. The pfmdr1 gene copy number was measured by real-time PCR as a ratio of the amount of pfmdr1 DNA to the amount of lactate dehydrogenase (ldh) DNA. Eight samples had pfmdr1 DNA/ldh DNA ratios ≥3. The isolates in all 8 samples fell into categories I and III and were significantly more resistant to mefloquine, quinine, artemisinin, and artesunate and more sensitive to chloroquine than the isolates in the 57 samples with <3 copies of the gene (P ≤ 0.001). Thus, measurement of pfmdr1 mutations and gene copy number may be useful for surveillance of mefloquine-resistant malaria in Southeast Asia.

scholarly journals EZH2 facilitates BMI1-dependent hepatocarcinogenesis through epigenetically silencing microRNA-200c

Oncogenesis ◽  
2020 ◽  
Vol 9 (11) ◽  
Author(s):  
Leibo Xu ◽  
Junlong Lin ◽  
Wanyu Deng ◽  
Weixin Luo ◽  
Yipei Huang ◽  
...  
Keyword(s):  
Copy Number ◽  
Gene Copy Number ◽  
Copy Number Gain ◽  
Gene Copy ◽  
Promoting Effect ◽  
Downstream Target ◽  
Bmi1 Expression ◽  
Hcc Cells

Abstract EZH2, a histone methyltransferase, has been shown to involve in cancer development and progression via epigenetic regulation of tumor suppressor microRNAs, whereas BMI1, a driver of hepatocellular carcinoma (HCC), is a downstream target of these microRNAs. However, it remains unclear whether EZH2 can epigenetically regulate microRNA expression to modulate BMI1-dependent hepatocarcinogenesis. Here, we established that high EZH2 expression correlated with enhanced tumor size, elevated metastasis, increased relapse, and poor prognosis in HCC patients. Further clinical studies revealed that EZH2 overexpression was positively correlated to its gene copy number gain/amplification in HCC. Mechanistically, EZH2 epigenetically suppressed miR-200c expression both in vitro and in vivo, and more importantly, miR-200c post-transcriptionally regulated BMI1 expression by binding to the 3′-UTR region of its mRNA. Furthermore, miR-200c overexpression inhibits the growth of HCC cells in vivo. Silencing miR-200c rescued the tumorigenicity of EZH2-depleted HCC cells, whereas knocking down BMI1 reduced the promoting effect of miR-200c depletion on HCC cell migration. Finally, combination treatment of EZH2 and BMI1 inhibitors further inhibited the viability of HCC cells compared with the cells treated with EZH2 or BMI1 inhibitor alone. Our findings demonstrated that alteration of EZH2 gene copy number status induced BMI1-mediated hepatocarcinogenesis via epigenetically silencing miR-200c, providing novel therapeutic targets for HCC treatment.

scholarly journals Increased activation product of complement 4 protein in plasma of individuals with schizophrenia

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Agnieszka Kalinowski ◽  
Joanna Liliental ◽  
Lauren A. Anker ◽  
Omer Linkovski ◽  
Collin Culbertson ◽  
...  
Keyword(s):  
Copy Number ◽  
Structural Variation ◽  
Gene Copy Number ◽  
Gene Copy ◽  
Activation Product ◽  
Protein Activation ◽  
Activation Products ◽  
Droplet Pcr ◽  
Complement 4

AbstractStructural variation in the complement 4 gene (C4) confers genetic risk for schizophrenia. The variation includes numbers of the increased C4A copy number, which predicts increased C4A mRNA expression. C4-anaphylatoxin (C4-ana) is a C4 protein fragment released upon C4 protein activation that has the potential to change the blood–brain barrier (BBB). We hypothesized that elevated plasma levels of C4-ana occur in individuals with schizophrenia (iSCZ). Blood was collected from 15 iSCZ with illness duration < 5 years and from 14 healthy controls (HC). Plasma C4-ana was measured by radioimmunoassay. Other complement activation products C3-ana, C5-ana, and terminal complement complex (TCC) were also measured. Digital-droplet PCR was used to determine C4 gene structural variation state. Recombinant C4-ana was added to primary brain endothelial cells (BEC) and permeability was measured in vitro. C4-ana concentration was elevated in plasma from iSCZ compared to HC (mean = 654 ± 16 ng/mL, 557 ± 94 respectively, p = 0.01). The patients also carried more copies of the C4AL gene and demonstrated a positive correlation between plasma C4-ana concentrations and C4A gene copy number. Furthermore, C4-ana increased the permeability of a monolayer of BEC in vitro. Our findings are consistent with a specific role for C4A protein in schizophrenia and raise the possibility that its activation product, C4-ana, increases BBB permeability. Exploratory analyses suggest the novel hypothesis that the relationship between C4-ana levels and C4A gene copy number could also be altered in iSCZ, suggesting an interaction with unknown genetic and/or environmental risk factors.

scholarly journals Leishmania naiffi and Leishmania guyanensis reference genomes highlight genome structure and gene evolution in the Viannia subgenus

2017 ◽  
Author(s):  
Simone Coughlan ◽  
Ali Shirley Taylor ◽  
Eoghan Feane ◽  
Mandy Sanders ◽  
Gabriele Schonian ◽  
...  
Keyword(s):  
Copy Number ◽  
Structural Changes ◽  
Gene Evolution ◽  
Genome Structure ◽  
Gene Copy Number ◽  
Gene Copy ◽  
Individual Species ◽  
Short Sequence ◽  
Origin And Evolution ◽  
Reference Genomes

AbstractThe unicellular protozoan parasite Leishmania causes the neglected tropical disease leishmaniasis, affecting 12 million people in 98 countries. In South America where the Viannia subgenus predominates, so far only L. (Viannia) braziliensis and L. (V.) panamensis have been sequenced, assembled and annotated as reference genomes. Addressing this deficit in molecular information can inform species typing, epidemiological monitoring and clinical treatment. Here, L. (V.) naiffi and L. (V.) guyanensis genomic DNA was sequenced to assemble these two genomes as draft references from short sequence reads. The methods used were tested using short sequence reads for L. braziliensis M2904 against its published reference as a comparison. This assembly and annotation pipeline identified 70 additional genes not annotated on the original M2904 reference. Phylogenetic and evolutionary comparisons of L. guyanensis and L. naiffi with ten other Viannia genomes revealed four traits common to all Viannia: aneuploidy, 22 orthologous groups of genes absent in other Leishmania subgenera, elevated TATE transposon copies, and a high NADH-dependent fumarate reductase gene copy number. Within the Viannia, there were limited structural changes in genome architecture specific to individual species: a 45 Kb amplification on chromosome 34 was present in all bar L. lainsoni, L. naiffi had a higher copy number of the virulence factor leishmanolysin, and laboratory isolate L. shawi M8408 had a possible minichromosome derived from the 3’ end of chromosome 34. This combination of genome assembly, phylogenetics and comparative analysis across an extended panel of diverse Viannia has uncovered new insights into the origin and evolution of this subgenus and can help improve diagnostics for leishmaniasis surveillance.

scholarly journals Estimating Copy-Number Proportions: The Comeback of Sanger Sequencing

Genes ◽  
2021 ◽  
Vol 12 (2) ◽  
pp. 283
Author(s):  
Eyal Seroussi
Keyword(s):  
Copy Number ◽  
Sanger Sequencing ◽  
Cytosine Methylation ◽  
Direct Sequencing ◽  
Information Source ◽  
Gene Copy Number ◽  
Cost Effective ◽  
Gene Copy ◽  
Base Editing ◽  
Recent Developments

Determination of the relative copy numbers of mixed molecular species in nucleic acid samples is often the objective of biological experiments, including Single-Nucleotide Polymorphism (SNP), indel and gene copy-number characterization, and quantification of CRISPR-Cas9 base editing, cytosine methylation, and RNA editing. Standard dye-terminator chromatograms are a widely accessible, cost-effective information source from which copy-number proportions can be inferred. However, the rate of incorporation of dye terminators is dependent on the dye type, the adjacent sequence string, and the secondary structure of the sequenced strand. These variable rates complicate inferences and have driven scientists to resort to complex and costly quantification methods. Because these complex methods introduce their own biases, researchers are rethinking whether rectifying distortions in sequencing trace files and using direct sequencing for quantification will enable comparable accurate assessment. Indeed, recent developments in software tools (e.g., TIDE, ICE, EditR, BEEP and BEAT) indicate that quantification based on direct Sanger sequencing is gaining in scientific acceptance. This commentary reviews the common obstacles in quantification and the latest insights and developments relevant to estimating copy-number proportions based on direct Sanger sequencing, concluding that bidirectional sequencing and sophisticated base calling are the keys to identifying and avoiding sequence distortions.

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