scholarly journals Genome analysis of the unicellular eukaryote Euplotes vannus reveals molecular basis for sex determination and tolerance to environmental stresses

2018 ◽  
Author(s):  
Xiao Chen ◽  
Yaohan Jiang ◽  
Feng Gao ◽  
Weibo Zheng ◽  
Timothy J. Krock ◽  
...  
Keyword(s):  
Sex Determination ◽  
Molecular Basis ◽  
De Novo ◽  
Stop Codon ◽  
Extreme Temperature ◽  
Environmental Stresses ◽  
Unicellular Eukaryote ◽  
Mating Types ◽  
Euplotes Vannus ◽  
Determination Mechanism

AbstractAs a model organism in studies of cell and environmental biology, the free-living and cosmopolitan ciliated protist Euplotes vannus has more than ten mating types (sexes) and shows strong resistance to environmental stresses. However, the molecular basis of its sex determination mechanism and how the cell responds to stress remain largely unknown. Here we report a combined analysis of de novo assembled high-quality macronucleus (MAC; i.e. somatic) genome and partial micronucleus (MIC; i.e. germline) genome of Euplotes vannus. Furthermore, MAC genomic and transcriptomic data from several mating types of E. vannus were investigated and gene expression levels were profiled under different environmental stresses, including nutrient scarcity, extreme temperature, salinity and the presence of free ammonia. We found that E. vannus, which possesses gene-sized nanochromosomes in its MAC, shares a similar pattern on frameshifting and stop codon usage as Euplotes octocarinatus and may be undergoing incipient sympatric speciation with Euplotes crassus. Somatic pheromone loci of E. vannus are generated from programmed DNA rearrangements of multiple germline macronuclear destined sequences (MDS) and the mating types of E. vannus are distinguished by the different combinations of pheromone loci instead of possessing mating type-specific genes. Lastly, we linked the resilience to environmental temperature change to the evolved loss of temperature stress-sensitive regulatory regions of HSP70 gene in E. vannus. Together, the genome resources generated in this study, which are available online at Euplotes vannus DB (http://evan.ciliate.org), provide new evidence for sex determination mechanism in eukaryotes and common pheromone-mediated cell-cell signaling and cross-mating.

scholarly journals In Vitro- and In Vivo-Generated Defective RNAs of Satellite Panicum Mosaic Virus Define cis-Acting RNA Elements Required for Replication and Movement

2000 ◽  
Vol 74 (5) ◽  
pp. 2247-2254 ◽  
Author(s):  
Wenping Qiu ◽  
Scholthof G. Karen-Beth
Keyword(s):  
Mosaic Virus ◽  
De Novo ◽  
Stop Codon ◽  
Dependent Manner ◽  
Defective Rnas ◽  
Systemic Spread ◽  
Rna Elements ◽  
Rna Domains ◽  
Rna Accumulation

ABSTRACT Satellite panicum mosaic virus (SPMV) depends on its helper virus, panicum mosaic virus (PMV), to provide trans-acting proteins for replication and movement. The 824-nucleotide (nt) genome of SPMV possesses an open reading frame encoding a 17.5-kDa capsid protein (CP), which is shown to be dispensable for SPMV replication. To localize cis-acting RNA elements required for replication and movement, a comprehensive set of SPMV cDNA deletion mutants was generated. The results showed that the 263-nt 3′ untranslated region (UTR) plus 73 nt upstream of the CP stop codon and the first 16 nt in the 5′ UTR are required for SPMV RNA amplification and/or systemic spread. A region from nt 17 to 67 within the 5′ UTR may have an accessory role in RNA accumulation, and a fragment bracketing nt 68 to 104 appears to be involved in the systemic movement of SPMV RNA in a host-dependent manner. Unexpectedly, defective RNAs (D-RNAs) accumulated de novo in millet plants coinfected with PMV and either of two SPMV mutants: SPMV-91, which is incapable of expressing the 17.5-kDa CP, and SPMV-GUG, which expresses low levels of the 17.5-kDa CP. The D-RNA derived from SPMV-91 was isolated from infected plants and used as a template to generate a cDNA clone. RNA transcripts derived from this 399-nt cDNA replicated and moved in millet plants coinoculated with PMV. The characterization of this D-RNA provided a biological confirmation that the critical RNA domains identified by the reverse genetic strategy are essential for SPMV replication and movement. The results additionally suggest that a potential “trigger” for spontaneous D-RNA accumulation may be associated with the absence or reduced accumulation of the 17.5-kDa SPMV CP. This represents the first report of a D-RNA associated with a satellite virus.

scholarly journals Translationskopplung via Termination-Reinitiation in Archaea und Bacteria

2021 ◽  
Author(s):  
◽  
Madeleine Huber
Keyword(s):  
Escherichia Coli ◽  
De Novo ◽  
Stop Codon ◽  
Haloferax Volcanii ◽  
Site Directed Mutagenesis ◽  
Start Codon ◽  
Directed Mutagenesis ◽  
Ribosome Display ◽  
E Coli

Operons wurden zuerst im Jahre 1961 beschrieben. Bis heute ist bekannt, dass die prokaryotischen Domänen Bacteria und Archaea Gene sowohl in monocistronischen als auch in bi- oder polycistronischen Transkripten exprimieren können. Häufig überlappen Gene sogar in ihren Sequenzen. Diese überlappenden Genpaare stehen nicht in Korrelation mit der Kompaktheit ihres Genoms. Das führt zu der Annahme, dass eine Art der Regulation vorliegt, welche weitere Proteine oder Gene nicht benötigt. Diese könnte eine gekoppelte Translation sein. Das bedeutet die Translation des stromabwärts-liegenden Gens ist abhängig von der Translation eines stromaufwärts-liegenden Gens. Diese Abhängigkeit kann zum Beispiel durch lang reichende Sekundärstrukturen entstehen, bei welchen Ribosomenbindestellen (RBS) des stromabwärts-liegenden Gens blockiert sind. Die de novo-Initiation am stromabwärts-liegenden Gen kann nur stattfinden, wenn das erste Gen translatiert wird und dabei die Sekundärstruktur an der RBS aufgeschmolzen wird. Für Genpaare in E. coli ist dieser Mechanismus gut untersucht. Ein anderes Beispiel für die Translationskopplung ist die Termination-Reinitiation, bei welcher ein Ribosom das erste Gen translatiert bis zum Stop-Codon, dort terminiert und direkt am stromabwärts-liegenden Start-Codon reinitiiert. Der Mechanismus via Termination-Reinitiation ist bis jetzt nur für eukaryontische Viren beschrieben worden. Im Gegensatz zu einer Kopplung über Sekundärstrukturen kommt es bei der Termination-Reinitiation am stromabwärts-liegenden Gen nicht zu einer de novo-Initiation sondern eine Reinitiation des Ribosoms findet statt. Diese Arbeit analysiert jene Art der Translationskopplung an Genen polycistronischer mRNAs in jeweils einem Modellorganismus als Vertreter der Archaea (Haloferax volcanii) und Bacteria (Escherichia coli). Hierfür wurden Reportergenvektoren erstellt, welche die überlappenden Genpaare an Reportergene fusionierten. Für diese Reportergene ist es möglich die Transkriptmenge zu quantifizieren sowie für die exprimierten Proteine Enzymassays durchgeführt werden können. Aus beiden Werten können Translationseffizienzen berechnet werden indem jeweils die Enzymaktivität pro Transkriptmenge ermittelt wird. Durch ein prämatures Stop-Codon in diesen Konstrukten ist es möglich zu unterscheiden ob es für die Translation des zweiten Gens essentiell ist, dass das Ribosom den Überlapp erreicht. Hiermit konnte für neun Genpaare in H. volcanii und vier Genpaare in E. coli gezeigt werden, dass eine Art der Kopplung stattfindet bei der es sich um eine Termination-Reinitiation handelt. Des Weiteren wurde analysiert, welche Auswirkungen intragene Shine-Dalgarno Sequenzen bei dem Event der Translationskopplung besitzen. Durch die Mutation solcher Motive und dem Vergleich der Translationseffizienzen der Konstrukte, mit und ohne einer SD Sequenz, wird für alle analysierten Genpaare beider Modellorganismen gezeigt, dass die SD Sequenz einen Einfluss auf diese Art der Kopplung hat. Zwischen den Genpaaren ist dieser Einfluss jedoch stark variabel. Weiterhin wurde der maximale Abstand zwischen zwei bicistronischen Genen untersucht, für welchen Translationskopplung via Termination-Reinitiation noch stattfinden kann. Hierfür wird durch site-directed mutagenesis jeweils ein prämatures Stop-Codon im stromaufwärts-liegenden Gen eingebracht, welches den intergenen Abstand zwischen den Genen in den jeweiligen Konstrukten vergrößert. Der Vergleich aller Konstrukte eines Genpaars zeigt in beiden Modellorganismen, dass die Termination-Reinitiation vom intergenen Abstand abhängig ist und die Translationseffizienz des stromabwärts-liegenden Reporters bereits ab 15 Nukleotiden Abstand abnimmt. Eine weitere Fragestellung dieser Arbeit war es, den genauen Mechanismus der Termination-Reinitiation zu analysieren. Für Ribosomen gibt es an der mRNA nach der Termination der Translation zwei Möglichkeiten: Entweder als 70S Ribosom bestehen zu bleiben und ein weiteres Start-Codon auf der mRNA zu suchen oder in seine beiden Untereinheiten zu dissoziieren, während die 50S Untereinheit die mRNA verlässt und die 30S Untereinheit über Wechselwirkungen an der mRNA verbleiben kann. Um diesen Mechanismus auf molekularer Ebene zu untersuchen, wird ein Versuchsablauf vorgestellt. Dieser ermöglicht das Event bei der Termination-Reinitiation in vitro zu analysieren. Eine Unterscheidung von 30S oder 70S Ribosomen bei der Reinitiation der Translation des stromabwärts-liegenden Gens wird ermöglicht. Die Idee dabei basiert auf einem ribosome display, bei welchem Translationskomplexe am Ende der Translation nicht in ihre Bestandteile zerfallen können, da die eingesetzte mRNA kein Stop-Codon enthält Der genaue Versuchsablauf, die benötigten Bestandteile sowie proof-of-principal Versuche sind in der Arbeit dargestellt und mögliche Optimierungen werden diskutiert.

scholarly journals Investigating the molecular basis of temperature‐dependent sex determination in the painted turtle

2010 ◽  
Vol 24 (S1) ◽  
Author(s):  
Katherine Lynn O'Shaughnessy ◽  
Nicole Valenzuela ◽  
Jennifer Neuwald ◽  
Robert Literman ◽  
Amanda Harris ◽  
...  
Keyword(s):  
Sex Determination ◽  
Molecular Basis ◽  
Painted Turtle ◽  
Temperature Dependent

scholarly journals The wild grape genome sequence provides insights into the transition from dioecy to hermaphroditism during grape domestication

2020 ◽  
Author(s):  
Hélène Badouin ◽  
Amandine Velt ◽  
François Gindraud ◽  
Timothée Flutre ◽  
Vincent Dumas ◽  
...  
Keyword(s):  
Vitis Vinifera ◽  
Sex Determination ◽  
Reference Genome ◽  
De Novo ◽  
Gene Content ◽  
Female Sterility ◽  
Genome Resequencing ◽  
Cultural Importance ◽  
Grape Genome ◽  
Whole Genome Resequencing

Grapevine has a major economical and cultural importance since antiquity. A key step in domestication was the transition from separate sexes (dioecy) in wild Vitis vinifera ssp. sylvestris (V. sylvestris) to hermaphroditism in cultivated Vitis vinifera ssp. vinifera. While the grapevine sex locus is known to be small, its precise boundaries, gene content and the sex-determining genes are unknown. Here we obtained a high-quality de novo reference genome for V. sylvestris and whole-genome resequencing data of a cross. Studying SNP segregation patterns, gene content and expression in wild and cultivated accessions allowed us to build a model for sex determination in grapevine. In this model, up- and down-regulation of a cytokinin regulator is sufficient to cause female sterility and reversal to hermaphroditism, respectively. This study highlights the importance of neo-functionalization of Y alleles in sex determination and provides a resource for studying genetic diversity in V. sylvestris and the genomic processes of grapevine domestication.

Key aspects of the primary sex determination mechanism are conserved across the genus Drosophila

Development ◽  
1998 ◽  
Vol 125 (16) ◽  
pp. 3259-3268 ◽  
Author(s):  
J.W. Erickson ◽  
T.W. Cline
Keyword(s):  
Sex Determination ◽  
Rapid Onset ◽  
Amino Acid Identity ◽  
Bzip Transcription Factor ◽  
Regulatory Sequence ◽  
Tightly Coupled ◽  
Pole Cells ◽  
High Level ◽  
Key Aspects ◽  
Determination Mechanism

In D. melanogaster, a set of ‘X:A numerator genes’, which includes sisterlessA (sisA), determines sex by controlling the transcription of Sex-lethal (Sxl). We characterized sisA from D. pseudoobscura and D. virilis and studied the timing of sisA and Sxl expression with single cell-cycle resolution in D. virilis, both to guide structure-function studies of sisA and to help understand sex determination evolution. We found that D. virilis sisA shares 58% amino acid identity with its melanogaster ortholog. The identities confirm sisA as an atypical bZIP transcription factor. Although virilis sisA can substitute for melanogaster sisA, the protein is not fully functional in a heterologous context. The putative sisA regulatory sequence CAGGTAG is a potential ‘numerator box,’ since it is shared with the other strong X:A numerator gene, sisB, and its target, SxlPe. Temporal and spatial features of sisA and SxlPe expression are strikingly conserved, including rapid onset and cessation of transcription in somatic nuclei, early cessation of sisA transcription in budding pole cells and persistent high-level sisA expression in yolk nuclei. Expression of sisA and Sxl is as tightly coupled in virilis as it is in melanogaster. Taken together, these data indicate that the same primary sex determination mechanism exists throughout the genus Drosophila.

Cross-sexual Transfer

2003 ◽  
Author(s):  
Mary Jane West-Eberhard
Keyword(s):  
Sex Determination ◽  
Sexually Dimorphic ◽  
Male And Female ◽  
Alternative Phenotypes ◽  
Opposite Sex ◽  
Critical Age ◽  
Sexual Traits ◽  
Discrete Traits ◽  
Dimorphic Species ◽  
Determination Mechanism

Distinctive male and female traits are perhaps the most familiar of all divergent specializations within species. In cross-sexual transfer, discrete traits that are expressed exclusively in one sex in an ancestral species appear in the opposite sex of descendants. An example is the expression of brood care by males in a lineage where ancestral females are the exclusive caretakers of the young, as in some voles (Thomas and Birney, 1979). Despite the prominence of sexual dimorphism and sex reversals in nature, and an early explicit treatment by Darwin, discussed in the next section, cross-sexual transfer is not often recognized as a major factor in the evolution of novelty (but see, on animals, Mayr, 1963, pp. 435-439; Mayr, 1970, p. 254; on plants, Iltis, 1983). When more widely investigated, cross-sexual transfer may prove to rival heterochrony and duplication as an important source of novelties in sexually dimorphic lineages. For this reason, I devote more attention here to cross-sexual transfer than to these other, well-established general patterns of change. The male and female of a sexually dimorphic species may be so different that it is easy to forget that each individual carries most or all of the genes necessary to produce the phenotype of the opposite sex. Sex determination, like caste determination and other switches between alternative phenotypes, depends on only a few genetic loci or, in many species, environmental factors (Bull, 1983). There is considerable flexibility in sex determination and facultative reversal in some taxa. Among fish, for example, there is even a species wherein sex is determined by juvenile size at a critical age (Francis and Barlow, 1993). The sex determination mechanism, whatever its nature, leads to a series of sex-limited responses, often coordinated by hormones and not necessarily all occurring at once. A distinguishing aspect of sexually dimorphic traits in adults is that there is often a close homology between the secondary sexual traits that are differently modified in the two sexes.

scholarly journals Molecular Basis for Natural Vegetative Propagation via Regeneration in North American Lake Cress, Rorippa aquatica (Brassicaceae)

2019 ◽  
Vol 61 (2) ◽  
pp. 353-369 ◽  
Author(s):  
Rumi Amano ◽  
Hokuto Nakayama ◽  
Risa Momoi ◽  
Emi Omata ◽  
Shizuka Gunji ◽  
...  
Keyword(s):  
Shoot Regeneration ◽  
Vegetative Propagation ◽  
Molecular Basis ◽  
Auxin Transport ◽  
Vascular Tissue ◽  
De Novo ◽  
Leaf Explants ◽  
Experimental Models ◽  
Polar Auxin Transport ◽  
Proximal Side

Abstract Some plant species have a striking capacity for regeneration in nature, including regeneration of the entire individual from explants. However, due to the lack of suitable experimental models, the regulatory mechanisms of spontaneous whole plant regeneration are mostly unknown. In this study, we established a novel model system to study these mechanisms using an amphibious plant within Brassicaceae, Rorippa aquatica, which naturally undergoes vegetative propagation via regeneration from leaf fragments. Morphological and anatomical observation showed that both de novo root and shoot organogenesis occurred from the proximal side of the cut edge transversely with leaf vascular tissue. Time-series RNA-seq analysis revealed that auxin and cytokinin responses were activated after leaf amputation and that regeneration-related genes were upregulated mainly on the proximal side of the leaf explants. Accordingly, we found that both auxin and cytokinin accumulated on the proximal side. Application of a polar auxin transport inhibitor retarded root and shoot regeneration, suggesting that the enhancement of auxin responses caused by polar auxin transport enhanced de novo organogenesis at the proximal wound site. Exogenous phytohormone and inhibitor applications further demonstrated that, in R. aquatica, both auxin and gibberellin are required for root regeneration, whereas cytokinin is important for shoot regeneration. Our results provide a molecular basis for vegetative propagation via de novo organogenesis.

scholarly journals Discovery of a New TLR Gene and Gene Expansion Event through Improved Desert Tortoise Genome Assembly with Chromosome-Scale Scaffolds

2020 ◽  
Vol 12 (2) ◽  
pp. 3917-3925
Author(s):  
Greer A Dolby ◽  
Matheo Morales ◽  
Timothy H Webster ◽  
Dale F DeNardo ◽  
Melissa A Wilson ◽  
...  
Keyword(s):  
Genome Assembly ◽  
Mojave Desert ◽  
De Novo ◽  
Stop Codon ◽  
Draft Genome ◽  
Gene Families ◽  
Sea Turtle ◽  
Desert Tortoise ◽  
Draft Genome Assembly ◽  
Gene Expansion

Abstract Toll-like receptors (TLRs) are a complex family of innate immune genes that are well characterized in mammals and birds but less well understood in nonavian sauropsids (reptiles). The advent of highly contiguous draft genomes of nonmodel organisms enables study of such gene families through analysis of synteny and sequence identity. Here, we analyze TLR genes from the genomes of 22 tetrapod species. Findings reveal a TLR8 gene expansion in crocodilians and turtles (TLR8B), and a second duplication (TLR8C) specifically within turtles, followed by pseudogenization of that gene in the nonfreshwater species (desert tortoise and green sea turtle). Additionally, the Mojave desert tortoise (Gopherus agassizii) has a stop codon in TLR8B (TLR8-1) that is polymorphic among conspecifics. Revised orthology further reveals a new TLR homolog, TLR21-like, which is exclusive to lizards, snakes, turtles, and crocodilians. These analyses were made possible by a new draft genome assembly of the desert tortoise (gopAga2.0), which used chromatin-based assembly to yield draft chromosomal scaffolds (L50 = 26 scaffolds, N50 = 28.36 Mb, longest scaffold = 107 Mb) and an enhanced de novo genome annotation with 25,469 genes. Our three-step approach to orthology curation and comparative analysis of TLR genes shows what new insights are possible using genome assemblies with chromosome-scale scaffolds that permit integration of synteny conservation data.

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