scholarly journals A toolkit enabling efficient, scalable and reproducible gene tagging in trypanosomatids

Open Biology ◽  
2015 ◽  
Vol 5 (1) ◽  
pp. 140197 ◽  
Author(s):  
Samuel Dean ◽  
Jack Sunter ◽  
Richard J. Wheeler ◽  
Ian Hodkinson ◽  
Eva Gluenz ◽  
...  
Keyword(s):  
Trypanosoma Brucei ◽  
Large Datasets ◽  
Gene Tagging ◽  
Deletion Mutants ◽  
Whole Genome ◽  
Leishmania Mexicana ◽  
Protein Coding ◽  
Achievable Goal ◽  
Endogenous Loci ◽  
Pcr Conditions

One of the first steps in understanding a protein's function is to determine its localization; however, the methods for localizing proteins in some systems have not kept pace with the developments in other fields, creating a bottleneck in the analysis of the large datasets that are generated in the post-genomic era. To address this, we developed tools for tagging proteins in trypanosomatids. We made a plasmid that, when coupled with long primer PCR, can be used to produce transgenes at their endogenous loci encoding proteins tagged at either terminus or within the protein coding sequence. This system can also be used to generate deletion mutants to investigate the function of different protein domains. We show that the length of homology required for successful integration precluded long primer PCR tagging in Leishmania mexicana . Hence, we developed plasmids and a fusion PCR approach to create gene tagging amplicons with sufficiently long homologous regions for targeted integration, suitable for use in trypanosomatids with less efficient homologous recombination than Trypanosoma brucei . Importantly, we have automated the primer design, developed universal PCR conditions and optimized the workflow to make this system reliable, efficient and scalable such that whole genome tagging is now an achievable goal.

scholarly journals Genome Analysis of Rhodococcus Sp. DSSKP-R-001: A Highly Effective β-Estradiol-Degrading Bacterium

2018 ◽  
Vol 2018 ◽  
pp. 1-11 ◽  
Author(s):  
Hongyan Zhao ◽  
Kejian Tian ◽  
Qing Qiu ◽  
Yu Wang ◽  
Hongyan Zhang ◽  
...  
Keyword(s):  
Sole Source ◽  
Rhodococcus Opacus ◽  
Whole Genome ◽  
Metabolic Potential ◽  
Protein Coding ◽  
Degrading Enzymes ◽  
Degrading Bacterium ◽  
Rhodococcus Jostii Rha1 ◽  
Multiple Strains ◽  
Rhodococcus Sp

We screened bacteria that use E2 as its sole source of carbon and energy for growth and identified them as Rhodococcus, and we named them DSSKP-R-001. For a better understanding of the metabolic potential of the strain, whole genome sequencing of Rhodococcus DSSKP-R-001 and annotation of the functional genes were performed. The genomic sketches included a predicted protein-coding gene of approximately 5.4 Mbp with G + C content of 68.72% and 5180. The genome of Rhodococcus strain DSSKP-R-001 consists of three replicons: one chromosome and two plasmids of 5.2, 0.09, and 0.09, respectively. The results showed that there were ten steroid-degrading enzymes distributed in the whole genome of the strain. The existence and expression of estradiol-degrading enzymes were verified by PCR and RTPCR. Finally, comparative genomics was used to compare multiple strains of Rhodococcus. It was found that Rhodococcus DSSKP-R-001 had the highest similarity to Rhodococcus sp. P14 and there were 2070 core genes shared with Rhodococcus sp. P14, Rhodococcus jostii RHA1, Rhodococcus opacus B4, and Rhodococcus equi 103S, showing evolutionary homology. In summary, this study provides a comprehensive understanding of the role of Rhodococcus DSSKP-R-001 in estradiol-efficient degradation of these assays for Rhodococcus. DSSKP-R-001 in bioremediation and evolution within Rhodococcus has important meaning.

scholarly journals Structural Elucidation and Cytotoxicity of a New 17-Membered Ring Lactone from Algerian Eryngium campestre

Molecules ◽  
2018 ◽  
Vol 23 (12) ◽  
pp. 3250 ◽  
Author(s):  
Ali Medbouhi ◽  
Aura Tintaru ◽  
Claire Beaufay ◽  
Jean-Valère Naubron ◽  
Nassim Djabou ◽  
...  
Keyword(s):  
Chemical Composition ◽  
Trypanosoma Brucei ◽  
Crude Extract ◽  
Membered Ring ◽  
Leishmania Mexicana ◽  
Similar Concentration ◽  
Trypanosoma Brucei Brucei ◽  
Aerial Parts ◽  
Germacrene D ◽  
Low Activity

The chemical composition of a hexanic extract of Eryngium campestre, obtained from its aerial parts, was investigated by GC-FID, GC/MS, HRMS, NMR and VCD analyses. The main compounds were germacrene D (23.6%), eudesma-4(15)-7-dien-1-β-ol (8.2%) and falcarindiol (9.4%), which are associated with a new uncommon and naturally found 17-membered ring lactone. This 17-membered ring features conjugated acetylenic bonds, named campestrolide (23.0%). The crude extract showed moderate antitrypanosomal (Trypanosoma brucei brucei), antileishmanial (Leishmania mexicana mexicana) and anticancer (cancerous macrophage-like murine cells) activities, and also displayed cytotoxicity, (human normal fibroblasts) in similar concentration ranges (IC50 = 3.0, 3.9, 4.0 and 4.4 µg/mL respectively). Likewise, campestrolide displayed low activity on all tested cells (IC50: 12.5–19.5 µM) except on Trypanosoma, on which it was very active and moderately selective (IC50 = 2.2 µM. SI= 8.9). In conclusion, the new compound that has been described, displaying a singular structure, possesses interesting antitrypanosomal activity that should be further investigated and improved.

scholarly journals Genomic Analysis of Sarcomyxa edulis Reveals the Basis of Its Medicinal Properties and Evolutionary Relationships

2021 ◽  
Vol 12 ◽  
Author(s):  
Fenghua Tian ◽  
Changtian Li ◽  
Yu Li
Keyword(s):  
Single Molecule ◽  
De Novo ◽  
Genomic Analysis ◽  
Single Copy ◽  
Whole Genome Sequence ◽  
Type I ◽  
Whole Genome ◽  
Uridine Diphosphate ◽  
Protein Coding ◽  
Medicinal Value

Yuanmo [Sarcomyxa edulis (Y.C. Dai, Niemelä & G.F. Qin) T. Saito, Tonouchi & T. Harada] is an important edible and medicinal mushroom endemic to Northeastern China. Here we report the de novo sequencing and assembly of the S. edulis genome using single-molecule real-time sequencing technology. The whole genome was approximately 35.65 Mb, with a G + C content of 48.31%. Genome assembly generated 41 contigs with an N50 length of 1,772,559 bp. The genome comprised 9,364 annotated protein-coding genes, many of which encoded enzymes involved in the modification, biosynthesis, and degradation of glycoconjugates and carbohydrates or enzymes predicted to be involved in the biosynthesis of secondary metabolites such as terpene, type I polyketide, siderophore, and fatty acids, which are responsible for the pharmacodynamic activities of S. edulis. We also identified genes encoding 1,3-β-glucan synthase and endo-1,3(4)-β-glucanase, which are involved in polysaccharide and uridine diphosphate glucose biosynthesis. Phylogenetic and comparative analyses of Basidiomycota fungi based on a single-copy orthologous protein indicated that the Sarcomyxa genus is an independent group that evolved from the Pleurotaceae family. The annotated whole-genome sequence of S. edulis can serve as a reference for investigations of bioactive compounds with medicinal value and the development and commercial production of superior S. edulis varieties.

scholarly journals Quorum sensing and genomic studies of a marine bacterium Vibrio variabilis strain T01

2015 ◽  
Author(s):  
Kok-Gan Chan ◽  
Nur Izzati Mohamad
Keyword(s):  
Quorum Sensing ◽  
Coastal Waters ◽  
Marine Bacterium ◽  
Cell Communication ◽  
Whole Genome ◽  
Whole Genome Analysis ◽  
Protein Coding ◽  
Signalling Molecule ◽  
Genomic Studies ◽  
Rna Genes

Vibrio variabilis strain T01 was isolated from the coastal waters in Hulu Selangor, Malaysia and its genome sequenced. This curved gram-negative bacterium shows cell-to-cell communication properties. The characteristics of the sequenced genome and its annotation processes are described here. The finished assembled whole genome of T01T exhibits genome size of 4,529,728 bp in 83 contigs with 46.22% G+C content, 4053 protein coding genes and 94 RNA genes. The whole genome analysis revealed the presence of quorum sensing signalling molecule synthase gene (luxM) which is crucial to understand the quorum sensing dependent phenotypes in this isolate.

Diverse patterns of expression of the cytochrome c oxidase subunit I gene and unassigned reading frames 4 and 5 during the life cycle of Trypanosoma brucei

1985 ◽  
Vol 5 (11) ◽  
pp. 3041-3047
Author(s):  
D P Jasmer ◽  
J E Feagin ◽  
K Stuart
Keyword(s):  
Life Cycle ◽  
Cytochrome C ◽  
Cytochrome C Oxidase ◽  
Trypanosoma Brucei ◽  
Protein Coding ◽  
Oxidase Subunit ◽  
Life Cycle Stages ◽  
Multiple Processes ◽  
Mitochondrial Transcripts ◽  
Reading Frames

Transcription of a maxicircle segment from Trypanosoma brucei 164 that contains nucleotide (nt) sequences corresponding to cytochrome c oxidase subunit I (COI) and unassigned reading frames (URFs) 4 and 5 of other mitochondrial systems was investigated. Two major transcripts that differ in size by ca. 200 nt map to each of the COI and URF4 genes, while a single major transcript maps to URF5. In total RNA, the larger COI transcript is more abundant in procyclic forms (PFs) than in bloodstream forms (BFs), the smaller COI and both URF4 transcripts have similar abundances in both forms, and the single URF5 transcript is more abundant in BF than PF. These patterns of expression differ in poly(A)+ RNA as a result of a higher proportion of poly(A)+ mitochondrial transcripts in PFs than in BFs. In addition, small (300- to 500-nt) RNAs that are transcribed from C-rich sequences located between putative protein-coding genes also exhibit diverse patterns of expression between life cycle stages and differences in polyadenylation in PFs compared with BFs. These observations suggest that multiple processes regulate the differential expression of mitochondrial genes in T. brucei.

scholarly journals Chromosome structure: DNA nucleotide sequence elements of a subset of the minichromosomes of the protozoan Trypanosoma brucei.

1991 ◽  
Vol 11 (8) ◽  
pp. 3823-3834 ◽  
Author(s):  
M Weiden ◽  
Y N Osheim ◽  
A L Beyer ◽  
L H Van der Ploeg
Keyword(s):  
Trypanosoma Brucei ◽  
Antigenic Variation ◽  
Microscopic Analysis ◽  
The Body ◽  
Surface Glycoprotein ◽  
Main Body ◽  
Electron Microscopic ◽  
Protein Coding ◽  
Cell Surface Glycoprotein ◽  
Telomere Repeat

The genome of the protozoan Trypanosoma brucei contains a set of about 100 minichromosomes of about 50 to 150 kb in size. The small size of these chromosomes, their involvement in antigenic variation, and their mitotic stability make them ideal candidates for a structural analysis of protozoan chromosomes and their telomeres. We show that a subset of the minichromosomes is composed predominantly of simple-sequence DNA, with over 90% of the length of the minichromosome consisting of a tandem array of 177-bp repeats, indicating that these molecules have limited protein-coding capacity. Proceeding from the tip of the telomere to a chromosome internal position, a subset of the minichromosomes contained the GGGTTA telomere repeat, a 29-bp telomere-derived repeat, a region containing 74-bp G + C-rich direct repeats separated by approximately 155 bp of A + T-rich DNA that has a bent character, and 50 to 150 kb of the 177-bp repeat. Several of the minichromosome-derived telomeres did not encode protein-coding genes, indicating that the repertoire of telomeric variant cell surface glycoprotein genes is restricted to some telomeres only. The telomere organization in trypanosomes shares striking similarities to the organization of telomeres and subtelomeres in humans, yeasts, and plasmodia. An electron microscopic analysis of the minichromosomes showed that they are linear molecules without abnormal structures in the main body of the chromosome. The structure of replicating molecules indicated that minichromosomes probably have a single bidirectional origin of replication located in the body of the chromosome. We propose a model for the structure of the trypanosome minichromosomes.

scholarly journals Glucose-6-Phosphate Dehydrogenase of Trypanosomatids: Characterization, Target Validation, and Drug Discovery

2011 ◽  
Vol 2011 ◽  
pp. 1-10 ◽  
Author(s):  
Shreedhara Gupta ◽  
Mariana Igoillo-Esteve ◽  
Paul A. M. Michels ◽  
Artur T. Cordeiro
Keyword(s):  
Oxidative Stress ◽  
Enzyme Activity ◽  
Trypanosoma Brucei ◽  
Redox State ◽  
Chemotherapeutic Agents ◽  
Dependent Manner ◽  
Target Validation ◽  
Leishmania Mexicana ◽  
State Dependent ◽  
Glucose 6 Phosphate Dehydrogenase

In trypanosomatids, glucose-6-phosphate dehydrogenase (G6PDH), the first enzyme of the pentosephosphate pathway, is essential for the defense of the parasite against oxidative stress. Trypanosoma brucei, Trypanosoma cruzi, and Leishmania mexicana G6PDHs have been characterized. The parasites' G6PDHs contain a unique 37 amino acid long N-terminal extension that in T. cruzi seems to regulate the enzyme activity in a redox-state-dependent manner. T. brucei and T. cruzi G6PDHs, but not their Leishmania spp. counterpart, are inhibited, in an uncompetitive way, by steroids such as dehydroepiandrosterone and derivatives. The Trypanosoma enzymes are more susceptible to inhibition by these compounds than the human G6PDH. The steroids also effectively kill cultured trypanosomes but not Leishmania and are presently considered as promising leads for the development of new parasite-selective chemotherapeutic agents.

scholarly journals Whole-Genome Sequence of SARS-CoV-2 Isolate Siena-1/2020

2020 ◽  
Vol 9 (39) ◽  
Author(s):  
Maria Grazia Cusi ◽  
David Pinzauti ◽  
Claudia Gandolfo ◽  
Gabriele Anichini ◽  
Gianni Pozzi ◽  
...  
Keyword(s):  
Amino Acid ◽  
Severe Acute Respiratory Syndrome ◽  
Genome Sequence ◽  
Amino Acid Change ◽  
Amplicon Sequencing ◽  
Spike Protein ◽  
Whole Genome Sequence ◽  
Whole Genome ◽  
Nanopore Sequencing ◽  
Protein Coding

ABSTRACT The complete genome sequence of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) isolate Siena-1/2020 was obtained by Nanopore sequencing, combining the direct RNA sequencing and amplicon sequencing approaches. The isolate belongs to the B1.1 lineage, which is prevalent in Europe, and contains a mutation in the spike protein coding sequence leading to the D614G amino acid change.

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