De Novo Transcriptome Meta-Assembly of the Mixotrophic Freshwater Microalga Euglena gracilis

Euglena gracilis is a well-known photosynthetic microeukaryote considered as the product of a secondary endosymbiosis between a green alga and a phagotrophic unicellular belonging to the same eukaryotic phylum as the parasitic trypanosomatids. As its nuclear genome has proven difficult to sequence, reliable transcriptomes are important for functional studies. In this work, we assembled a new consensus transcriptome by combining sequencing reads from five independent studies. Based on a detailed comparison with two previously released transcriptomes, our consensus transcriptome appears to be the most complete so far. Remapping the reads on it allowed us to compare the expression of the transcripts across multiple culture conditions at once and to infer a functionally annotated network of co-expressed genes. Although the emergence of meaningful gene clusters indicates that some biological signal lies in gene expression levels, our analyses confirm that gene regulation in euglenozoans is not primarily controlled at the transcriptional level. Regarding the origin of E. gracilis, we observe a heavily mixed gene ancestry, as previously reported, and rule out sequence contamination as a possible explanation for these observations. Instead, they indicate that this complex alga has evolved through a convoluted process involving much more than two partners.

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Insights into the Genome Sequence ofChromobacterium amazonenseIsolated from a Tropical Freshwater Lake

International Journal of Genomics ◽

10.1155/2018/1062716 ◽

2018 ◽

Vol 2018 ◽

pp. 1-10

Author(s):

Alexandre Bueno Santos ◽

Patrícia Silva Costa ◽

Anderson Oliveira do Carmo ◽

Gabriel da Rocha Fernandes ◽

Larissa Lopes Silva Scholte ◽

...

Keyword(s):

De Novo ◽

Genomic Diversity ◽

Protein Coding ◽

Biotechnological Potential ◽

Draft Assembly ◽

Functional Studies ◽

Alpha Hemolysin ◽

Type Iv ◽

Nudix Hydrolases ◽

Colicin V

Members of the genusChromobacteriumhave been isolated from geographically diverse ecosystems and exhibit considerable metabolic flexibility, as well as biotechnological and pathogenic properties in some species. This study reports the draft assembly and detailed sequence analysis ofChromobacterium amazonensestrain 56AF. The de novo-assembled genome is 4,556,707 bp in size and contains 4294 protein-coding and 95 RNA genes, including 88 tRNA, six rRNA, and one tmRNA operon. A repertoire of genes implicated in virulence, for example, hemolysin, hemolytic enterotoxins, colicin V, lytic proteins, and Nudix hydrolases, is present. The genome also contains a collection of genes of biotechnological interest, including esterases, lipase, auxins, chitinases, phytoene synthase and phytoene desaturase, polyhydroxyalkanoates, violacein, plastocyanin/azurin, and detoxifying compounds. Importantly, unlike otherChromobacteriumspecies, the 56AF genome contains genes for pore-forming toxin alpha-hemolysin, a type IV secretion system, among others. The analysis of theC. amazonensestrain 56AF genome reveals the versatility, adaptability, and biotechnological potential of this bacterium. This study provides molecular information that may pave the way for further comparative genomics and functional studies involvingChromobacterium-related isolates and improves our understanding of the global genomic diversity ofChromobacteriumspecies.

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Transcriptional regulation of Pseudomonas aeruginosa rhlR, encoding a quorum-sensing regulatory protein

Microbiology ◽

10.1099/mic.0.26282-0 ◽

2003 ◽

Vol 149 (11) ◽

pp. 3073-3081 ◽

Cited By ~ 87

Author(s):

Gerardo Medina ◽

Katy Juárez ◽

Rafael Díaz ◽

Gloria Soberón-Chávez

Keyword(s):

Pseudomonas Aeruginosa ◽

Transcriptional Regulation ◽

Quorum Sensing ◽

Regulatory Protein ◽

Culture Conditions ◽

Transcriptional Level ◽

Coding Region ◽

Upstream Gene ◽

Transcription Start Sites ◽

Response Systems

The Pseudomonas aeruginosa rhlR gene encodes the transcriptional regulator RhlR which has a central role in the quorum-sensing response. Different gene products involved in bacterial pathogenesis are regulated at the transcriptional level by two quorum-sensing response systems, Las and Rhl. The expression of rhlR has been reported to be under the control of the Las system, but its transcriptional regulation has not been studied in detail. Here, the rhlR promoter region has been characterized and shown to present four different transcription start sites, two of which are included in the upstream gene (rhlB) coding region. It was found that rhlR expression is not only dependent on LasR but also on different regulatory proteins such as Vfr and RhlR itself, and also on the alternative sigma factor σ 54. It is reported that rhlR expression is partially LasR-independent under certain culture conditions and is strongly influenced by environmental factors.

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Steroidogenic Factor-1 (SF-1)-Driven Differentiation of Murine Embryonic Stem (ES) Cells into a Gonadal Lineage

Endocrinology ◽

10.1210/en.2011-0219 ◽

2011 ◽

Vol 152 (7) ◽

pp. 2870-2882 ◽

Cited By ~ 24

Author(s):

Unmesh Jadhav ◽

J. Larry Jameson

Keyword(s):

Target Genes ◽

De Novo ◽

Embryoid Body ◽

Es Cells ◽

Embryonic Stem ◽

Cell Lineage ◽

Culture Conditions ◽

Steroidogenic Factor 1 ◽

Lineage Differentiation ◽

And Function

Steroidogenic factor 1 (SF-1) is essential for the development and function of steroidogenic tissues. Stable incorporation of SF-1 into embryonic stem cells (SF-1-ES cells) has been shown to prime the cells for steroidogenesis. When provided with exogenous cholesterol substrate, and after treatment with retinoic acid and cAMP, SF-1-ES cells produce progesterone but do not produce other steroids such as cortisol, estradiol, or testosterone. In this study, we explored culture conditions that optimize SF-1-mediated differentiation of ES cells into defined steroidogenic lineages. When embryoid body formation was used to facilitate cell lineage differentiation, SF-1-ES cells were found to be restricted in their differentiation, with fewer cells entering neuronal pathways and a larger fraction entering the steroidogenic lineage. Among the differentiation protocols tested, leukemia inhibitory factor (LIF) removal, followed by prolonged cAMP treatment was most efficacious for inducing steroidogenesis in SF-1-ES cells. In this protocol, a subset of SF-1-ES cells survives after LIF withdrawal, undergoes morphologic differentiation, and recovers proliferative capacity. These cells are characterized by induction of steroidogenic enzyme genes, use of de novo cholesterol, and production of multiple steroids including estradiol and testosterone. Microarray studies identified additional pathways associated with SF-1 mediated differentiation. Using biotinylated SF-1 in chromatin immunoprecipitation assays, SF-1 was shown to bind directly to multiple target genes, with induction of binding to some targets after steroidogenic treatment. These studies indicate that SF-1 expression, followed by LIF removal and treatment with cAMP drives ES cells into a steroidogenic pathway characteristic of gonadal steroid-producing cells.

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Transcriptional regulation of the human CAD gene during myeloid differentiation

Molecular and Cellular Biology ◽

10.1128/mcb.7.5.1961-1966.1987 ◽

1987 ◽

Vol 7 (5) ◽

pp. 1961-1966

Author(s):

G N Rao ◽

E S Buford ◽

J N Davidson

Keyword(s):

Gene Expression ◽

De Novo ◽

Morphological Changes ◽

Transcriptional Level ◽

Carbamoyl Phosphate ◽

Aspartate Transcarbamylase ◽

Trans Retinoic Acid ◽

Beta Actin ◽

Cad Gene ◽

Cad Protein

CAD codes for a trifunctional protein involved in the catalysis of the first three enzymatic activities in the de novo pyrimidine biosynthetic pathway, namely, carbamoyl-phosphate synthetase II (EC 6.3.5.5), aspartate transcarbamylase (EC 2.1.3.2), and dihydroorotase (EC 3.5.2.3). CAD regulation was studied in the human promyelocyte leukemic line HL-60 as it differentiated into monocytic or granulocytic lineages after induction by 12-O-tetradecanoylphorbol-13-acetate or trans-retinoic acid and dibutyryl cyclic AMP, respectively. Within 12 h of induction of HL-60 cells with either inducer, total cellular levels of CAD RNA essentially disappeared. On the other hand, no apparent decreases in beta-actin RNA levels were seen even 48 h after HL-60 cells were induced, as compared with untreated cells. With nuclear runoff assays, it was clearly shown that the inactivation of CAD gene expression during the induction of HL-60 cells with either inducer was at the transcriptional level. The nuclear runoff experiments also demonstrated that the CAD gene expression was shut down in less than 4 h after induction, well before morphological changes were observed in these cells. At the enzymatic level, the activity of aspartate transcarbamylase, one of the three enzymes encoded by the CAD gene, decreased by about half within 24 h of induction, suggesting a CAD protein half-life of 24 h in differentiating HL-60 cells. Nevertheless, this means that significant levels of aspartate transcarbamylase activity remained even after the cells have stopped proliferating. From the RNA data, it is clear that CAD gene expression is rapidly turned off as promyelocytes begin to terminally differentiate into macrophages and granulocytes. We suspect that the inactivation of the CAD gene in induced HL-60 cells is a consequence of the differentiating cells leaving the cell cycle and becoming nonproliferating.

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CNNM2-Related Disorders: Phenotype and Its Severity Were Associated With the Mode of Inheritance

Frontiers in Pediatrics ◽

10.3389/fped.2021.699568 ◽

2021 ◽

Vol 9 ◽

Author(s):

Han Zhang ◽

Ye Wu ◽

Yuwu Jiang

Keyword(s):

De Novo ◽

Serum Magnesium ◽

Brain Magnetic Resonance Imaging ◽

Functional Studies ◽

Pathogenic Variants ◽

Significant Difference ◽

Domain Variations ◽

Type 3

CNNM2 (Cystathionine-β-synthase-pair Domain Divalent Metal Cation Transport Mediator 2) pathogenic variants have been reported to cause hypomagnesemia, epilepsy, and intellectual disability/developmental delay (ID/DD). We identified two new cases with CNNM2 novel de novo pathogenic variants, c.814T>C and c.976G>C. They both presented with infantile-onset epilepsy with DD and hypomagnesemia refractory to magnesium supplementation. To date, 21 cases with CNNM2-related disorders have been reported. We combined all 23 cases to analyze the features of CNNM2-related disorders. The phenotypes can be classified into three types: type 1, autosomal dominant (AD) inherited simple hypomagnesemia; type 2, AD inherited hypomagnesemia with epilepsy and ID/DD; and type 3, autosomal recessive (AR) inherited hypomagnesemia with epilepsy and ID/DD. All five type 1 cases had no epilepsy or ID/DD; they all had hypomagnesemia, and three of them presented with symptoms secondary to hypomagnesemia. Fifteen type 2 patients could have ID/DD and seizures, which can be controlled with antiseizure medications (ASMs); their variations clustered in the DUF21 domain of CNNM2. All three type 3 patients had seizures from 1 to 6 days after birth; the seizures were refractory, and 1/3 had status epilepticus; ID/DD in these AR-inherited cases was more severe than that of AD-inherited cases; they all had abnormalities of brain magnetic resonance imaging (MRI). Except for one patient whose serum magnesium was the lower limit of normal, others had definite hypomagnesemia. Hypomagnesemia could be improved after magnesium supplement but could not return to the normal level. Variations in the CBS2 domain may be related to lower serum magnesium. However, there was no significant difference in the level of serum magnesium among the patients with three different types of CNNM2-related disorders. The severity of different phenotypes was therefore not explained by decreased serum magnesium. We expanded the spectrum of CNNM2 variants and classified the phenotypes of CNNM2-related disorders into three types. We found that DUF21 domain variations were most associated with CNNM2-related central nervous system phenotypes, whereas hypomagnesemia was more pronounced in patients with CBS2 domain variations, and AR-inherited CNNM2-related disorders had the most severe phenotype. These results provide important clues for further functional studies of CNNM2 and provide basic foundations for more accurate genetic counseling.

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Direct estimate of the spontaneous mutation rate uncovers the effects of drift and recombination in theChlamydomonas reinhardtiiplastid genome

10.1101/031898 ◽

2015 ◽

Author(s):

Rob W Ness ◽

Susanne A Kraemer ◽

Nick Colegrave ◽

Peter D Keightley

Keyword(s):

Mutation Rate ◽

Genetic Drift ◽

Plastid Genome ◽

De Novo ◽

Spontaneous Mutation ◽

Genome Structure ◽

Nuclear Genome ◽

De Novo Mutation ◽

Direct Estimate ◽

Plastid Genomes

Plastids perform crucial cellular functions, including photosynthesis, across a wide variety of eukaryotes. Since endosymbiosis, plastids have maintained independent genomes that now display a wide diversity of gene content, genome structure, gene regulation mechanisms, and transmission modes. The evolution of plastid genomes depends on an input ofde novomutation, but our knowledge of mutation in the plastid is limited to indirect inference from patterns of DNA divergence between species. Here, we use a mutation accumulation experiment, where selection acting on mutations is rendered ineffective, combined with whole-plastid genome sequencing to directly characterize de novo mutation inChlamydomonas reinhardtii. We show that the mutation rates of the plastid and nuclear genomes are similar, but that the base spectra of mutations differ significantly. We integrate our measure of the mutation rate with a population genomic dataset of 20 individuals, and show that the plastid genome is subject to substantially stronger genetic drift than the nuclear genome. We also show that high levels of linkage disequilibrium in the plastid genome are not due to restricted recombination, but are instead a consequence of increased genetic drift. One likely explanation for increased drift in the plastid genome is that there are stronger effects of genetic hitchhiking. The presence of recombination in the plastid is consistent with laboratory studies inC. reinhardtiiand demonstrates that although the plastid genome is thought to be uniparentally inherited, it recombines in nature at a rate similar to the nuclear genome.

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gcaPDA: A Haplotype-resolved Diploid Assembler

10.1101/2021.05.31.446328 ◽

2021 ◽

Author(s):

Xie Min ◽

Linfeng Yang ◽

Chenglin Jiang ◽

Shenshen Wu ◽

Cheng Luo ◽

...

Keyword(s):

De Novo ◽

Low Complexity ◽

F1 Hybrid ◽

Functional Studies ◽

Assembly Result ◽

Eukaryotic Genomes

Generating chromosome-scale haplotype resolved assembly is important for functional studies. However, current de novo assemblers are either haploid assemblers that discard allelic information, or diploid assemblers that can only tackle genomes of low complexity. Here, we report a diploid assembler, gcaPDA (gamete cells assisted Phased Diploid Assembler), which exploits haploid gamete cells to assist in resolving haplotypes. We generate chromosome-scale phased diploid assemblies for the highly heterozygous and repetitive genome of a maize F1 hybrid using gcaPDA and evaluate the assembly result thoroughly. With applicability of coping with complex genomes and fewer restrictions on application than other diploid assemblers, gcaPDA is likely to find broad applications in studies of eukaryotic genomes.

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Regulation and a possible stage-specific function of Oct-2 during pre-B-cell differentiation.

Molecular and Cellular Biology ◽

10.1128/mcb.11.10.4885 ◽

1991 ◽

Vol 11 (10) ◽

pp. 4885-4894 ◽

Cited By ~ 23

Author(s):

C L Miller ◽

A L Feldhaus ◽

J W Rooney ◽

L D Rhodes ◽

C H Sibley ◽

...

Keyword(s):

B Cell ◽

Transforming Growth Factor Beta ◽

Transforming Growth Factor ◽

De Novo ◽

Interleukin 1 ◽

Transcriptional Level ◽

Immunoglobulin Gene ◽

B Cell Differentiation ◽

Nf Kappa B ◽

Specific Expression

The Oct-2 gene appears to encode a developmental regulator of immunoglobulin gene transcription. We demonstrate that the Oct-2 gene is expressed at low levels in a variety of transformed pre-B-cell lines and is induced specifically in these cells by lipopolysaccharide signalling. This work extends an earlier observation in the pre-B-cell line 70Z/3 and therefore suggests that the inducible expression of the Oct-2 gene, like that of the kappa gene, is a characteristic feature of the pre-B stage of B-cell development. In 70Z/3 cells, the lymphokine interleukin-1 also induces the expression of the Oct-2 and kappa loci. Interestingly, expression of the Oct-2 gene is rapidly induced at the transcriptional level and may not require de novo protein synthesis. Since the changes in the activity of the Oct-2 locus completely correlate with the changes of the activity of the kappa locus, the two genes may be transcriptionally regulated by a common trans-acting factor. In 70Z/3 cells, transforming growth factor beta, an inhibitor of kappa-gene induction, blocks the upregulation of Oct-2 but not the activation of NF-kappa B. These results suggest that the combinatorial action of increased levels of Oct-2 and activated NF-kappa B may be necessary for the proper stage-specific expression of the kappa locus.

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Site-specific methylation of adenine in the nuclear genome of a eucaryote, Tetrahymena thermophila.

Molecular and Cellular Biology ◽

10.1128/mcb.6.7.2364 ◽

1986 ◽

Vol 6 (7) ◽

pp. 2364-2370 ◽

Cited By ~ 23

Author(s):

G S Harrison ◽

R C Findly ◽

K M Karrer

Keyword(s):

Heat Shock ◽

Protein Gene ◽

De Novo ◽

Tetrahymena Thermophila ◽

Nuclear Genome ◽

Histone H4 ◽

Ciliated Protozoan ◽

Site Specific ◽

Logarithmic Growth ◽

I Gene

DNA in the polyploid macronucleus of the ciliated protozoan Tetrahymena thermophila contains the modified base N6-methyladenine. We identified two GATC sites which are methylated in most or all of the 45 copies of the macronuclear genome. One site is 2 kilobases 5' to the histone H4-I gene, and the other is 5 kilobases 3' to the 73-kilodalton heat shock protein gene. These sites are de novo methylated between 10 and 16 h after initiation of conjugation, during macronuclear anlage development. The methylation states of these two GATC sites and four other unmethylated GATC sites do not change in the DNA of cells cultured under conditions which change the activity of the genes, including logarithmic growth, starvation, and heat shock.

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