scholarly journals Genetic Basis of De Novo Appearance of Carotenoid Ornamentation in Bare-Parts of Canaries

2019 ◽  
Author(s):  
Małgorzata Anna Gazda ◽  
Matthew B. Toomey ◽  
Pedro M. Araújo ◽  
Ricardo J. Lopes ◽  
Sandra Afonso ◽  
...  
Keyword(s):  
Genetic Basis ◽  
De Novo ◽  
Bird Species ◽  
Beta Carotene ◽  
Full Length ◽  
Nonsynonymous Mutation ◽  
Retinal Tissue ◽  
Cleavage Activity ◽  
Causative Locus

ABSTRACTUnlike wild and domestic canaries (Serinus canaria), or any of the three dozen species of finches in genus Serinus, the domestic urucum breed of canaries exhibits bright red bills and legs. This novel bare-part coloration offers a unique opportunity to understand how leg and bill coloration evolve in birds. To identify the causative locus, we resequenced the genome of urucum canaries and performed a range of analyses to search for genotype-to-phenotype associations across the genome. We identified a nonsynonymous mutation in the gene BCO2 (beta-carotene oxygenase 2, also known as BCDO2), an enzyme involved in the cleavage and breakdown of full-length carotenoids into short apocarotenoids. Protein structural models and in vitro functional assays indicate that the urucum mutation abrogates the carotenoid cleavage activity of BCO2. Consistent with the predicted loss of carotenoid cleavage activity, urucum canaries had increased levels of full-length carotenoid pigments in bill tissue and a significant reduction in levels of carotenoid cleavage products (apocarotenoids) in retinal tissue compared to other breeds of canaries. We hypothesize that carotenoid-based bare-part coloration might be readily gained, modified, or lost through simple switches in the enzymatic activity or regulation of BCO2 and this gene may be an important mediator in the evolution of bare-part coloration among bird species.

scholarly journals Genetic Basis of De Novo Appearance of Carotenoid Ornamentation in Bare Parts of Canaries

2020 ◽  
Vol 37 (5) ◽  
pp. 1317-1328 ◽  
Author(s):  
Małgorzata Anna Gazda ◽  
Matthew B Toomey ◽  
Pedro M Araújo ◽  
Ricardo J Lopes ◽  
Sandra Afonso ◽  
...  
Keyword(s):  
Genetic Basis ◽  
De Novo ◽  
Bird Species ◽  
Beta Carotene ◽  
Full Length ◽  
Nonsynonymous Mutation ◽  
Retinal Tissue ◽  
Cleavage Activity ◽  
Important Mediator

Abstract Unlike wild and domestic canaries (Serinus canaria), or any of the three dozen species of finches in genus Serinus, the domestic urucum breed of canaries exhibits bright red bills and legs. This novel trait offers a unique opportunity to understand the mechanisms of bare-part coloration in birds. To identify the mutation producing the colorful phenotype, we resequenced the genome of urucum canaries and performed a range of analyses to search for genotype-to-phenotype associations across the genome. We identified a nonsynonymous mutation in the gene BCO2 (beta-carotene oxygenase 2, also known as BCDO2), an enzyme involved in the cleavage and breakdown of full-length carotenoids into short apocarotenoids. Protein structural models and in vitro functional assays indicate that the urucum mutation abrogates the carotenoid-cleavage activity of BCO2. Consistent with the predicted loss of carotenoid-cleavage activity, urucum canaries tended to have increased levels of full-length carotenoid pigments in bill tissue and reduced levels of carotenoid-cleavage products (apocarotenoids) in retinal tissue compared with other breeds of canaries. We hypothesize that carotenoid-based bare-part coloration might be readily gained, modified, or lost through simple switches in the enzymatic activity or regulation of BCO2 and this gene may be an important mediator in the evolution of bare-part coloration among bird species.

scholarly journals Perturbation in the Conserved Methyltransferase-Polymerase Interface of Flavivirus NS5 Differentially Affects Polymerase Initiation and Elongation

2014 ◽  
Vol 89 (1) ◽  
pp. 249-261 ◽  
Author(s):  
Jiqin Wu ◽  
Guoliang Lu ◽  
Bo Zhang ◽  
Peng Gong
Keyword(s):  
Crystal Structure ◽  
Rna Polymerase ◽  
Rna Synthesis ◽  
De Novo ◽  
Full Length ◽  
Intramolecular Interactions ◽  
Rna Dependent Rna Polymerase ◽  
Hydrophobic Residues ◽  
Functional Relevance

ABSTRACTThe flavivirus NS5 is a natural fusion of a methyltransferase (MTase) and an RNA-dependent RNA polymerase (RdRP). Analogous to DNA-dependent RNA polymerases, the NS5 polymerase initiates RNA synthesis through ade novomechanism and then makes a transition to a processive elongation phase. However, whether and how the MTase affects polymerase activities through intramolecular interactions remain elusive. By solving the crystal structure of the Japanese encephalitis virus (JEV) NS5, we recently identified an MTase-RdRP interface containing a set of six hydrophobic residues highly conserved among flaviviruses. To dissect the functional relevance of this interface, we made a series of JEV NS5 constructs with mutations of these hydrophobic residues and/or with the N-terminal first 261 residues and other residues up to the first 303 residues deleted. Compared to the wild-type (WT) NS5, full-length NS5 variants exhibited consistent up- or downregulation of the initiation activities in two types of polymerase assays. Five representative full-length NS5 constructs were then tested in an elongation assay, from which the apparent single-nucleotide incorporation rate constant was estimated. Interestingly, two constructs exhibited different elongation kinetics from the WT NS5, with an effect rather opposite to what was observed at initiation. Moreover, constructs with MTase and/or the linker region (residues 266 to 275) removed still retained polymerase activities, albeit at overall lower levels. However, further removal of the N-terminal extension (residues 276 to 303) abolished regular template-directed synthesis. Together, our data showed that the MTase-RdRP interface is relevant in both polymerase initiation and elongation, likely with different regulation mechanisms in these two major phases of RNA synthesis.IMPORTANCEThe flavivirus NS5 is very unique in having a methyltransferase (MTase) placed on the immediate N terminus of its RNA-dependent RNA polymerase (RdRP). We recently solved the crystal structure of the full-length NS5, which revealed a conserved interface between MTase and RdRP. Building on this discovery, here we carried outin vitropolymerase assays to address the functional relevance of the interface interactions. By explicitly probing polymerase initiation and elongation activities, we found that perturbation in the MTase-RdRP interface had different impacts on different phases of synthesis, suggesting that the roles and contribution of the interface interactions may change upon phase transitions. By comparing the N-terminal-truncated enzymes with the full-length NS5, we collected data to indicate the indispensability to regular polymerase activities of a region that was functionally unclarified previously. Taken together, we provide biochemical evidence and mechanistic insights for the cross talk between the two enzyme modules of flavivirus NS5.

scholarly journals Comparative Genomics and Full-Length TprK Profiling of Treponema pallidum subsp. pallidum Reinfection

2019 ◽  
Author(s):  
Amin Addetia ◽  
Lauren C. Tantalo ◽  
Michelle J. Lin ◽  
Hong Xie ◽  
Meei-Li Huang ◽  
...  
Keyword(s):  
Outer Membrane ◽  
Causative Agent ◽  
Genetic Basis ◽  
Antigenic Variation ◽  
Treponema Pallidum ◽  
Variable Region ◽  
Full Length ◽  
Comparative Genomic ◽  
Definition Of

AbstractDeveloping a vaccine against Treponema pallidum subspecies pallidum, the causative agent of syphilis, remains a public health priority. Syphilis vaccine design efforts have been complicated by lack of an in vitro T. pallidum culture system, prolific antigenic variation in outer membrane protein TprK, and lack of functional annotation for nearly half of the genes. Understanding the genetic basis of T. pallidum reinfection can provide insights into variation among strains that escape cross-protective immunity. Here, we present comparative genomic sequencing and deep, full-length tprK profiling of two T. pallidum isolates from blood from the same patient that were collected six years apart. Notably, this patient was diagnosed with syphilis four times, with two of these episodes meeting the definition of neurosyphilis, during this interval. Outside of the highly variable tprK gene, we identified 14 coding changes in 13 genes. Nine of these genes putatively localized to the periplasmic or outer membrane spaces, consistent with a potential role in serological immunoevasion. Using a newly developed full-length tprK deep sequencing protocol, we profiled the diversity of this gene that far outpaces the rest of the genome. Intriguingly, we found that the reinfecting isolate demonstrated less diversity across each tprK variable region compared to the isolate from the first infection. Notably, the two isolates did not share any full-length TprK sequences. Our results are consistent with an immunodominant-evasion model in which the diversity of TprK explains the ability of T. pallidum to successfully reinfect individuals, even when they have been infected with the organism multiple times.Author SummaryThe causative agent of syphilis, Treponema pallidum subspecies pallidum, is capable of repeat infections in people, suggesting that the human immune response does not develop sufficiently broad or long-lasting immunity to cover treponemal diversity. Here, we examined the genomes from two blood-derived isolates of T. pallidum derived 6 years apart from a patient who had syphilis four times during the same period to understand the genetic basis of reinfection. We found a paucity of coding changes across the genome outside of the highly variable tprK gene. Using deep profiling of the full-length tprK gene, we found surprisingly that the two isolates did not share any full-length TprK sequences.

In Situ Localization of PPAR Gamma and Uncoupling Protein in Mouse Embryo Sections Using Digoxigenin-Labeled Riboprobes

1996 ◽  
Vol 54 ◽  
pp. 32-33
Author(s):  
C. Jennermann ◽  
S. A. Kliewer ◽  
D. C. Morris
Keyword(s):  
Alkaline Phosphatase ◽  
Room Temperature ◽  
Uncoupling Protein ◽  
Ppar Gamma ◽  
Nuclear Hormone Receptor ◽  
Full Length ◽  
Northern Analysis ◽  
Peroxisome Proliferator

Peroxisome proliferator-activated receptor gamma (PPARg) is a member of the nuclear hormone receptor superfamily and has been shown in vitro to regulate genes involved in lipid metabolism and adipocyte differentiation. By Northern analysis, we and other researchers have shown that expression of this receptor predominates in adipose tissue in adult mice, and appears first in whole-embryo mRNA at 13.5 days postconception. In situ hybridization was used to find out in which developing tissues PPARg is specifically expressed.Digoxigenin-labeled riboprobes were generated using the Genius™ 4 RNA Labeling Kit from Boehringer Mannheim. Full length PPAR gamma, obtained by PCR from mouse liver cDNA, was inserted into pBluescript SK and used as template for the transcription reaction. Probes of average size 200 base pairs were made by partial alkaline hydrolysis of the full length transcripts. The in situ hybridization assays were performed as described previously with some modifications. Frozen sections (10 μm thick) of day 18 mouse embryos were cut, fixed with 4% paraformaldehyde and acetylated with 0.25% acetic anhydride in 1.0M triethanolamine buffer. The sections were incubated for 2 hours at room temperature in pre-hybridization buffer, and were then hybridized with a probe concentration of 200μg per ml at 70° C, overnight in a humidified chamber. Following stringent washes in SSC buffers, the immunological detection steps were performed at room temperature. The alkaline phosphatase labeled, anti-digoxigenin antibody and detection buffers were purchased from Boehringer Mannheim. The sections were treated with a blocking buffer for one hour and incubated with antibody solution at a 1:5000 dilution for 2 hours, both at room temperature. Colored precipitate was formed by exposure to the alkaline phosphatase substrate nitrobluetetrazoliumchloride/ bromo-chloroindlylphosphate.

Einfluß der Glucose auf die [14C] Thymidin-Einbaurate von Ehrlich-Ascitescarcinomzellen in vitro

1969 ◽  
Vol 08 (02) ◽  
pp. 196-206 ◽  
Author(s):  
Dieter. Kummer
Keyword(s):  
De Novo

ZusammenfassungIn nahezu glucosefreier Suspension von Ehrlich-Ascitescarcinomzellen bewirkt die Zufuhr von Glucose 2,5 × 10–4 bis 10–2 M:1. Hemmung der [14C] Thymidin-Einbaurate in die Zellen.2. Aktivierung des Ribonucleotid-Reductase-Systems und damit Stimulierung der Desoxyribonucleotidsynthese (auch der Thymidintriphosphat-de-novo-Synthese).3. Blockierung der Thymidinkinase über Endprodukthemmung, wodurch die Minderung des [14C] Thymidin-Einbaus in die Zellen erklärbar ist.

Особенности морфогенеза редкого вида Allium neriniflorum (Herb.) Backer в условиях in vitro

2019 ◽  
pp. 37-41 ◽  
Author(s):  
Альбина Шамсуновна Ахметова ◽  
Альфия Ануровна Зарипова
Keyword(s):  
De Novo

Показана возможность эффективного применения метода культуры тканей для размножения Allium neriniflorum (Herb.) Backer. Исследуемый вид является декоративным растением, размножение которого затруднено из-за низкой всхожести семян и ослабленной способности к формированию дочерних луковиц. Разработана технология клонального микроразмножения из стерильных луковиц. В качестве исходного материала использовали семена A. neriniflorum. Подобраны условия стерилизации, позволяющие достичь максимального числа (75 %) жизнеспособных эксплантов. Поверхностную стерилизацию проводили в ламинар-боксе с использованием в качестве стерилизующего агента 0,1 % раствор диацида. Семена сначала обрабатывали 70 % этанолом, затем стерилизующим раствором. Экспозиция стерилизующих растворов составляла от 5 до 9 мин. Показано, что способность к индуцированному морфогенезу существенно зависит от состава питательной среды. Максимальное число луковиц образовывалось на среде QL — 9 шт./эксплант. Исследуемые виды обладали высокой способностью к мультипликации и формированию полноценных растений при подобранных условиях культивирования in vitro. Выявленная морфогенетическая активность зачаточного побега, сегментов чешуй и донца стерильной луковицы A. neriniflorum, проявляющаяся в способности регенерировать побеги de novo, что возможно только в культуре in vitro, обеспечивает формирование полноценных луковиц. Луковицы, полученные in vitro, включали в последующие циклы микроразмножения. Культура тканей и органов in vitro позволяет размножать A. neriniflorum с более высоким коэффициентом размножения. От одной стерильной луковицы можно получить до 7000 луковиц в год. При традиционном вегетативном способе размножения материнская луковица формирует 1, редко 2 дочерние луковицы.

Study of Phosphocalcic Glasses from SiO2-CaO-P2O5 System with and Without Silver II. The bioactivity analysis by FTIR, SEM methods and microbiological study of silver-doped glasses

2017 ◽  
Vol 68 (6) ◽  
pp. 1188-1192
Author(s):  
Daniela Avram ◽  
Nicolae Angelescu ◽  
Dan Nicolae Ungureanu ◽  
Ionica Ionita ◽  
Iulian Bancuta ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Body Fluid ◽  
Pathogenic Bacteria ◽  
De Novo ◽  
Microbiological Examination ◽  
Doped Glasses ◽  
P2o5 System ◽  
Scanning Electron

The study in vitro of the glass powders bioactivity was performed by soaking them in simulated body fluid for 3 to 21 days at a temperature of 37�C and pH = 7.20. The synthesis de novo of hydroxyapatite, post soaking was confirmed by Fourier Transform Infrared spectroscopy (FTIR) and scanning electron microscopy (SEM). The study of the antimicrobial activity was performed by microbiological examination on two strains of pathogenic bacteria involved in postoperative nosocomial infections.

scholarly journals Stereotypic neutralizing VH antibodies against SARS-CoV-2 spike protein receptor binding domain in COVID-19 patients and healthy individuals

2021 ◽  
pp. eabd6990
Author(s):  
Sang Il Kim ◽  
Jinsung Noh ◽  
Sujeong Kim ◽  
Younggeun Choi ◽  
Duck Kyun Yoo ◽  
...  
Keyword(s):  
B Cells ◽  
Receptor Binding ◽  
Neutralizing Antibodies ◽  
De Novo ◽  
Binding Domain ◽  
Host Cells ◽  
Spike Protein ◽  
Receptor Binding Domain ◽  
Healthy Individuals

Stereotypic antibody clonotypes exist in healthy individuals and may provide protective immunity against viral infections by neutralization. We observed that 13 out of 17 patients with COVID-19 had stereotypic variable heavy chain (VH) antibody clonotypes directed against the receptor-binding domain (RBD) of SARS-CoV-2 spike protein. These antibody clonotypes were comprised of immunoglobulin heavy variable (IGHV)3-53 or IGHV3-66 and immunoglobulin heavy joining (IGHJ)6 genes. These clonotypes included IgM, IgG3, IgG1, IgA1, IgG2, and IgA2 subtypes and had minimal somatic mutations, which suggested swift class switching after SARS-CoV-2 infection. The different immunoglobulin heavy variable chains were paired with diverse light chains resulting in binding to the RBD of SARS-CoV-2 spike protein. Human antibodies specific for the RBD can neutralize SARS-CoV-2 by inhibiting entry into host cells. We observed that one of these stereotypic neutralizing antibodies could inhibit viral replication in vitro using a clinical isolate of SARS-CoV-2. We also found that these VH clonotypes existed in six out of 10 healthy individuals, with IgM isotypes predominating. These findings suggest that stereotypic clonotypes can develop de novo from naïve B cells and not from memory B cells established from prior exposure to similar viruses. The expeditious and stereotypic expansion of these clonotypes may have occurred in patients infected with SARS-CoV-2 because they were already present.

scholarly journals Identification of Promiscuous African Swine Fever Virus T-Cell Determinants Using a Multiple Technical Approach

Vaccines ◽  
2021 ◽  
Vol 9 (1) ◽  
pp. 29
Author(s):  
Laia Bosch-Camós ◽  
Elisabet López ◽  
María Jesús Navas ◽  
Sonia Pina-Pedrero ◽  
Francesc Accensi ◽  
...  
Keyword(s):  
T Cell ◽  
Mononuclear Cells ◽  
African Swine Fever Virus ◽  
African Swine Fever ◽  
Cd8 T Cell ◽  
Protective Role ◽  
Full Length ◽  
T Cell Epitopes ◽  
Peripheral Blood Mononuclear

The development of subunit vaccines against African swine fever (ASF) is mainly hindered by the lack of knowledge regarding the specific ASF virus (ASFV) antigens involved in protection. As a good example, the identity of ASFV-specific CD8+ T-cell determinants remains largely unknown, despite their protective role being established a long time ago. Aiming to identify them, we implemented the IFNγ ELISpot as readout assay, using as effector cells peripheral blood mononuclear cells (PBMCs) from pigs surviving experimental challenge with Georgia2007/1. As stimuli for the ELISpot, ASFV-specific peptides or full-length proteins identified by three complementary strategies were used. In silico prediction of specific CD8+ T-cell epitopes allowed identifying a 19-mer peptide from MGF100-1L, as frequently recognized by surviving pigs. Complementarily, the repertoire of SLA I-bound peptides identified in ASFV-infected porcine alveolar macrophages (PAMs), allowed the characterization of five additional SLA I-restricted ASFV-specific epitopes. Finally, in vitro stimulation studies using fibroblasts transfected with plasmids encoding full-length ASFV proteins, led to the identification of MGF505-7R, A238L and MGF100-1L as promiscuously recognized antigens. Interestingly, each one of these proteins contain individual peptides recognized by surviving pigs. Identification of the same ASFV determinants by means of such different approaches reinforce the results presented here.

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