scholarly journals Resurrecting ancestral genes in bacteria to interpret ancient biosignatures

2017 ◽  
Vol 375 (2109) ◽  
pp. 20160352 ◽  
Author(s):  
Betul Kacar ◽  
Lionel Guy ◽  
Eric Smith ◽  
John Baross
Keyword(s):  
Carbonic Anhydrase ◽  
Single Molecule ◽  
Single Gene ◽  
Molecular Systems ◽  
Protein Functions ◽  
Beta Carbonic Anhydrase ◽  
Sequence Reconstruction ◽  
Living Organisms ◽  
Geologic Record ◽  
Molecular Components

Two datasets, the geologic record and the genetic content of extant organisms, provide complementary insights into the history of how key molecular components have shaped or driven global environmental and macroevolutionary trends. Changes in global physico-chemical modes over time are thought to be a consistent feature of this relationship between Earth and life, as life is thought to have been optimizing protein functions for the entirety of its approximately 3.8 billion years of history on the Earth. Organismal survival depends on how well critical genetic and metabolic components can adapt to their environments, reflecting an ability to optimize efficiently to changing conditions. The geologic record provides an array of biologically independent indicators of macroscale atmospheric and oceanic composition, but provides little in the way of the exact behaviour of the molecular components that influenced the compositions of these reservoirs. By reconstructing sequences of proteins that might have been present in ancient organisms, we can downselect to a subset of possible sequences that may have been optimized to these ancient environmental conditions. How can one use modern life to reconstruct ancestral behaviours? Configurations of ancient sequences can be inferred from the diversity of extant sequences, and then resurrected in the laboratory to ascertain their biochemical attributes. One way to augment sequence-based, single-gene methods to obtain a richer and more reliable picture of the deep past, is to resurrect inferred ancestral protein sequences in living organisms, where their phenotypes can be exposed in a complex molecular-systems context, and then to link consequences of those phenotypes to biosignatures that were preserved in the independent historical repository of the geological record. As a first step beyond single-molecule reconstruction to the study of functional molecular systems, we present here the ancestral sequence reconstruction of the beta-carbonic anhydrase protein. We assess how carbonic anhydrase proteins meet our selection criteria for reconstructing ancient biosignatures in the laboratory, which we term palaeophenotype reconstruction. This article is part of the themed issue ‘Reconceptualizing the origins of life’.

scholarly journals Resurrecting ancestral genes in bacteria to interpret ancient biosignatures

2017 ◽  
Author(s):  
Betul Kacar ◽  
Lionel Guy ◽  
Eric Smith ◽  
John Baross
Keyword(s):  
Carbonic Anhydrase ◽  
Single Molecule ◽  
Single Gene ◽  
Molecular Systems ◽  
Protein Functions ◽  
Beta Carbonic Anhydrase ◽  
Sequence Reconstruction ◽  
Living Organisms ◽  
Geologic Record ◽  
Molecular Components

SummaryTwo datasets, the geologic record and the genetic content of extant organisms, provide complementary insights into the history of how key molecular components have shaped or driven global environmental and macroevolutionary trends. Changes in global physicochemical modes over time are thought to be a consistent feature of this relationship between Earth and life, as life is thought to have been optimizing protein functions for the entirety of its ∼3.8 billion years of history on Earth. Organismal survival depends on how well critical genetic and metabolic components can adapt to their environments, reflecting an ability to optimize efficiently to changing conditions. The geologic record provides an array of biologically independent indicators of macroscale atmospheric and oceanic composition, but provides little in the way of the exact behavior of the molecular components that influenced the compositions of these reservoirs. By reconstructing sequences of proteins that might have been present in ancient organisms, we can identify a subset of possible sequences that may have been optimized to these ancient environmental conditions. How can extant life be used to reconstruct ancestral phenotypes? Configurations of ancient sequences can be inferred from the diversity of extant sequences, and then resurrected in the lab to ascertain their biochemical attributes. One way to augment sequence-based, single-gene methods to obtain a richer and more reliable picture of the deep past, is to resurrect inferred ancestral protein sequences in living organisms, where their phenotypes can be exposed in a complex molecular-systems context, and to then link consequences of those phenotypes to biosignatures that were preserved in the independent historical repository of the geological record. As a first-step beyond single molecule reconstruction to the study of functional molecular systems, we present here the ancestral sequence reconstruction of the beta-carbonic anhydrase protein. We assess how carbonic anhydrase proteins meet our selection criteria for reconstructing ancient biosignatures in the lab, which we term paleophenotype reconstruction.

scholarly journals Live-cell imaging reveals the spatiotemporal organization of endogenous RNA polymerase II phosphorylation at a single gene

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Linda S. Forero-Quintero ◽  
William Raymond ◽  
Tetsuya Handa ◽  
Matthew N. Saxton ◽  
Tatsuya Morisaki ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Single Molecule ◽  
Computational Models ◽  
Spatial Organization ◽  
Fluorescent Antibody ◽  
Single Gene ◽  
Single Copy ◽  
Living Cells ◽  
Live Cell

AbstractThe carboxyl-terminal domain of RNA polymerase II (RNAP2) is phosphorylated during transcription in eukaryotic cells. While residue-specific phosphorylation has been mapped with exquisite spatial resolution along the 1D genome in a population of fixed cells using immunoprecipitation-based assays, the timing, kinetics, and spatial organization of phosphorylation along a single-copy gene have not yet been measured in living cells. Here, we achieve this by combining multi-color, single-molecule microscopy with fluorescent antibody-based probes that specifically bind to different phosphorylated forms of endogenous RNAP2 in living cells. Applying this methodology to a single-copy HIV-1 reporter gene provides live-cell evidence for heterogeneity in the distribution of RNAP2 along the length of the gene as well as Serine 5 phosphorylated RNAP2 clusters that remain separated in both space and time from nascent mRNA synthesis. Computational models determine that 5 to 40 RNAP2 cluster around the promoter during a typical transcriptional burst, with most phosphorylated at Serine 5 within 6 seconds of arrival and roughly half escaping the promoter in ~1.5 minutes. Taken together, our data provide live-cell support for the notion of efficient transcription clusters that transiently form around promoters and contain high concentrations of RNAP2 phosphorylated at Serine 5.

scholarly journals Direct observation of frequency modulated transcription in single cells using light activation

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Daniel R Larson ◽  
Christoph Fritzsch ◽  
Liang Sun ◽  
Xiuhau Meng ◽  
David S Lawrence ◽  
...  
Keyword(s):  
Single Cell ◽  
Single Molecule ◽  
Single Cell Analysis ◽  
Cell Model ◽  
Single Cells ◽  
Single Gene ◽  
Gene Activation ◽  
Light Activation ◽  
Dynamic Synthesis ◽  
Single Cell Model

Single-cell analysis has revealed that transcription is dynamic and stochastic, but tools are lacking that can determine the mechanism operating at a single gene. Here we utilize single-molecule observations of RNA in fixed and living cells to develop a single-cell model of steroid-receptor mediated gene activation. We determine that steroids drive mRNA synthesis by frequency modulation of transcription. This digital behavior in single cells gives rise to the well-known analog dose response across the population. To test this model, we developed a light-activation technology to turn on a single steroid-responsive gene and follow dynamic synthesis of RNA from the activated locus.

scholarly journals Single Molecule Techniques Can Distinguish the Photophysical Processes Governing Metal-Enhanced Fluorescence

2022 ◽  
Author(s):  
Stefania Impellizzeri ◽  
Gregory J, Hodgson ◽  
Nicholas P. Dogantzis
Keyword(s):  
Single Molecule ◽  
Organic Molecules ◽  
Near Field ◽  
Comprehensive Understanding ◽  
Enhanced Fluorescence ◽  
Metal Enhanced Fluorescence ◽  
Maximum Enhancement ◽  
Molecular Systems ◽  
Photophysical Processes ◽  
Underlying Processes

<p>Plasmonic metal nanoparticles can impact the behaviour of organic molecules in a number of ways, including enhancing or quenching fluorescence. Only through a comprehensive understanding of the fundamental photophysical processes regulating nano-molecular interactions can these effects be controlled, and exploited to the fullest extent possible. Metal-enhanced fluorescence (MEF) is governed by two underlying processes, increased rate of fluorophore excitation and increased fluorophore emission, the balance between which has implications for optimizing hybrid nanoparticle-molecular systems for various applications. We report groundbreaking work on the use of single molecule fluorescence microscopy to distinguish between the two mechanistic components of MEF, in a model system consisting of two analogous boron dipyrromethene (BODIPY) fluorophores and triangular silver nanoparticles (AgNP). We demonstrate that the increased excitation MEF mechanism occurs to approximately the same extent for both dyes, but that the BODIPY with the higher quantum yield of fluorescence experiences a greater degree of MEF via the increased fluorophore emission mechanism, and higher overall enhancement, as a result of its superior ability to undergo near-field interactions with AgNP. We foresee that this knowledge and methodology will be used to tailor MEF to meet the needs of different applications, such as those requiring maximum enhancement of fluorescence intensity or instead prioritizing excited-state photochemistry. </p>

scholarly journals Applications of molecular networks in biomedicine

2019 ◽  
Vol 4 (1) ◽  
Author(s):  
Monica Chagoyen ◽  
Juan A G Ranea ◽  
Florencio Pazos
Keyword(s):  
Single Gene ◽  
Point Of View ◽  
Molecular Networks ◽  
Large Sets ◽  
Software Packages ◽  
Number Of Genes ◽  
Complex Phenomena ◽  
Network Approaches ◽  
Molecular Components ◽  
Standard Software

Abstract Due to the large interdependence between the molecular components of living systems, many phenomena, including those related to pathologies, cannot be explained in terms of a single gene or a small number of genes. Molecular networks, representing different types of relationships between molecular entities, embody these large sets of interdependences in a framework that allow their mining from a systemic point of view to obtain information. These networks, often generated from high-throughput omics datasets, are used to study the complex phenomena of human pathologies from a systemic point of view. Complementing the reductionist approach of molecular biology, based on the detailed study of a small number of genes, systemic approaches to human diseases consider that these are better reflected in large and intricate networks of relationships between genes. These networks, and not the single genes, provide both better markers for diagnosing diseases and targets for treating them. Network approaches are being used to gain insight into the molecular basis of complex diseases and interpret the large datasets associated with them, such as genomic variants. Network formalism is also suitable for integrating large, heterogeneous and multilevel datasets associated with diseases from the molecular level to organismal and epidemiological scales. Many of these approaches are available to nonexpert users through standard software packages.

scholarly journals An Inactivation Switch Enables Rhythms in a Neurospora Clock Model

2019 ◽  
Vol 20 (12) ◽  
pp. 2985 ◽  
Author(s):  
Abhishek Upadhyay ◽  
Michael Brunner ◽  
Hanspeter Herzel
Keyword(s):  
Feedback Loop ◽  
Design Principle ◽  
Temporal Dynamics ◽  
Model Organism ◽  
Transcription Activator ◽  
White Collar Complex ◽  
Living Organisms ◽  
Underlying Mechanisms ◽  
Molecular Components ◽  
Novel Model

Autonomous endogenous time-keeping is ubiquitous across many living organisms, known as the circadian clock when it has a period of about 24 h. Interestingly, the fundamental design principle with a network of interconnected negative and positive feedback loops is conserved through evolution, although the molecular components differ. Filamentous fungus Neurospora crassa is a well-established chrono-genetics model organism to investigate the underlying mechanisms. The core negative feedback loop of the clock of Neurospora is composed of the transcription activator White Collar Complex (WCC) (heterodimer of WC1 and WC2) and the inhibitory element called FFC complex, which is made of FRQ (Frequency protein), FRH (Frequency interacting RNA Helicase) and CK1a (Casein kinase 1a). While exploring their temporal dynamics, we investigate how limit cycle oscillations arise and how molecular switches support self-sustained rhythms. We develop a mathematical model of 10 variables with 26 parameters to understand the interactions and feedback among WC1 and FFC elements in nuclear and cytoplasmic compartments. We performed control and bifurcation analysis to show that our novel model produces robust oscillations with a wild-type period of 22.5 h. Our model reveals a switch between WC1-induced transcription and FFC-assisted inactivation of WC1. Using the new model, we also study the possible mechanisms of glucose compensation. A fairly simple model with just three nonlinearities helps to elucidate clock dynamics, revealing a mechanism of rhythms’ production. The model can further be utilized to study entrainment and temperature compensation.

Beta-Carbonic Anhydrase 1 from Trichomonas Vaginalis as New Antiprotozoan Drug Target

2021 ◽  
Author(s):  
Claudiu T. Supuran ◽  
Anna Di Fiore ◽  
Seppo Parkkila ◽  
Giuseppina De Simone
Keyword(s):  
Carbonic Anhydrase ◽  
Drug Target ◽  
Trichomonas Vaginalis ◽  
Beta Carbonic Anhydrase ◽  
Carbonic Anhydrase 1

BRCA Testing by Single-Molecule Molecular Inversion Probes

2017 ◽  
Vol 63 (2) ◽  
pp. 503-512 ◽  
Author(s):  
Kornelia Neveling ◽  
Arjen R Mensenkamp ◽  
Ronny Derks ◽  
Michael Kwint ◽  
Hicham Ouchene ◽  
...  
Keyword(s):  
Genetic Testing ◽  
Copy Number Variation ◽  
Single Molecule ◽  
Copy Number ◽  
Single Gene ◽  
Work Flow ◽  
Analytical Sensitivity ◽  
Brca1 And Brca2 ◽  
Number Variation ◽  
Molecular Inversion Probes

Abstract BACKGROUND Despite advances in next generation DNA sequencing (NGS), NGS-based single gene tests for diagnostic purposes require improvements in terms of completeness, quality, speed, and cost. Single-molecule molecular inversion probes (smMIPs) are a technology with unrealized potential in the area of clinical genetic testing. In this proof-of-concept study, we selected 2 frequently requested gene tests, those for the breast cancer genes BRCA1 and BRCA2, and developed an automated work flow based on smMIPs. METHODS The BRCA1 and BRCA2 smMIPs were validated using 166 human genomic DNA samples with known variant status. A generic automated work flow was built to perform smMIP-based enrichment and sequencing for BRCA1, BRCA2, and the checkpoint kinase 2 (CHEK2) c.1100del variant. RESULTS Pathogenic and benign variants were analyzed in a subset of 152 previously BRCA-genotyped samples, yielding an analytical sensitivity and specificity of 100%. Following automation, blind analysis of 65 in-house samples and 267 Norwegian samples correctly identified all true-positive variants (&gt;3000), with no false positives. Consequent to process optimization, turnaround times were reduced by 60% to currently 10–15 days. Copy number variants were detected with an analytical sensitivity of 100% and an analytical specificity of 88%. CONCLUSIONS smMIP-based genetic testing enables automated and reliable analysis of the coding sequences of BRCA1 and BRCA2. The use of single-molecule tags, double-tiled targeted enrichment, and capturing and sequencing in duplo, in combination with automated library preparation and data analysis, results in a robust process and reduces routine turnaround times. Furthermore, smMIP-based copy number variation analysis could make independent copy number variation tools like multiplex ligation-dependent probes amplification dispensable.

scholarly journals Biochemical and structural characterisation of a protozoan beta-carbonic anhydrase from Trichomonas vaginalis

2020 ◽  
Vol 35 (1) ◽  
pp. 1292-1299 ◽  
Author(s):  
Linda J. Urbański ◽  
Anna Di Fiore ◽  
Latifeh Azizi ◽  
Vesa P. Hytönen ◽  
Marianne Kuuslahti ◽  
...  
Keyword(s):  
Carbonic Anhydrase ◽  
Trichomonas Vaginalis ◽  
Structural Characterisation ◽  
Beta Carbonic Anhydrase

scholarly journals Haemolytic actinoporins interact with carbohydrates using their lipid-binding module

2017 ◽  
Vol 372 (1726) ◽  
pp. 20160216 ◽  
Author(s):  
Koji Tanaka ◽  
Jose M. M. Caaveiro ◽  
Koldo Morante ◽  
Kouhei Tsumoto
Keyword(s):  
Protein Interactions ◽  
Pore Formation ◽  
Plasma Membranes ◽  
Lipid Binding ◽  
Target Cells ◽  
Sea Anemones ◽  
Molecular Systems ◽  
Membrane Pores ◽  
Living Organisms ◽  
Lipid Protein Interactions

Pore-forming toxins (PFTs) are proteins endowed with metamorphic properties that enable them to stably fold in water solutions as well as in cellular membranes. PFTs produce lytic pores on the plasma membranes of target cells conducive to lesions, playing key roles in the defensive and offensive molecular systems of living organisms. Actinoporins are a family of potent haemolytic toxins produced by sea anemones vigorously studied as a paradigm of α-helical PFTs, in the context of lipid–protein interactions, and in connection with nanopore technologies. We have recently reported that fragaceatoxin C (FraC), an actinoporin, engages biological membranes with a large adhesive motif allowing the simultaneous attachment of up to four lipid molecules prior to pore formation. Since actinoporins also interact with carbohydrates, we sought to understand the molecular and energetic basis of glycan recognition by FraC. By employing structural and biophysical methodologies, we show that FraC engages glycans with low affinity using its lipid-binding module. Contrary to other PFTs requiring separate domains for glycan and lipid recognition, the small single-domain actinoporins economize resources by achieving dual recognition with a single binding module. This mechanism could enhance the recruitment of actinoporins to the surface of target tissues in their marine environment. This article is part of the themed issue ‘Membrane pores: from structure and assembly, to medicine and technology’.

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