scholarly journals Constitutive expression, not a particular primary sequence, is the important feature of the H3 replacement variant hv2 in Tetrahymena thermophila.

1997 ◽  
Vol 17 (11) ◽  
pp. 6303-6310 ◽  
Author(s):  
L Yu ◽  
M A Gorovsky
Keyword(s):  
Protein Sequence ◽  
Tetrahymena Thermophila ◽  
Histone H3 ◽  
Constitutive Expression ◽  
Histone Variants ◽  
Wild Type ◽  
Ciliated Protozoan ◽  
Knockout Mutant ◽  
Double Knockout ◽  
Constitutive Synthesis

Although quantitatively minor replication-independent (replacement) histone variants have been found in a wide variety of organisms, their functions remain unknown. Like the H3.3 replacement variants of vertebrates, hv2, an H3 variant in the ciliated protozoan Tetrahymena thermophila, is synthesized and deposited in nuclei of nongrowing cells. Although hv2 is clearly an H3.3-like replacement variant by its expression, sequence analysis indicates that it evolved independently of the H3.3 variants of multicellular eukaryotes. This suggested that it is the constitutive synthesis, not the particular protein sequence, of these variants that is important in the function of H3 replacement variants. Here, we demonstrate that the gene (HHT3) encoding hv2 or either gene (HHT1 or HHT2) encoding the abundant major H3 can be completely knocked out in Tetrahymena. Surprisingly, when cells lacking hv2 are starved, a major histone H3 mRNA transcribed by the HHT2 gene, which is synthesized little, if at all, in wild-type nongrowing cells, is easily detectable. Both HHT2 and HHT3 knockout strains show no obvious defect during vegetative growth. In addition, a mutant with the double knockout of HHT1 and HHT3 is viable while the HHT2 HHT3 double-knockout mutant is not. These results argue strongly that cells require a constitutively expressed H3 gene but that the particular sequence being expressed is not critical.

scholarly journals Sphingolipid C-9 Methyltransferases Are Important for Growth and Virulence but Not for Sensitivity to Antifungal Plant Defensins in Fusarium graminearum

2008 ◽  
Vol 8 (2) ◽  
pp. 217-229 ◽  
Author(s):  
Vellaisamy Ramamoorthy ◽  
Edgar B. Cahoon ◽  
Mercy Thokala ◽  
Jagdeep Kaur ◽  
Jia Li ◽  
...  
Keyword(s):  
Fusarium Graminearum ◽  
Type Strain ◽  
Wild Type Strain ◽  
Fungal Growth ◽  
Wild Type ◽  
Knockout Mutant ◽  
Double Knockout ◽  
Disease Symptoms ◽  
Plant Defensins ◽  
Antifungal Action

ABSTRACT The C-9-methylated glucosylceramides (GlcCers) are sphingolipids unique to fungi. They play important roles in fungal growth and pathogenesis, and they act as receptors for some antifungal plant defensins. We have identified two genes, FgMT1 and FgMT2, that each encode a putative sphingolipid C-9 methyltransferase (C-9-MT) in the fungal pathogen Fusarium graminearum and complement a Pichia pastoris C-9-MT-null mutant. The ΔFgmt1 mutant produced C-9-methylated GlcCer like the wild-type strain, PH-1, whereas the ΔFgmt2 mutant produced 65 to 75% nonmethylated and 25 to 35% methylated GlcCer. No ΔFgmt1ΔFgmt2 double-knockout mutant producing only nonmethylated GlcCer could be recovered, suggesting that perhaps C-9-MTs are essential in this pathogen. This is in contrast to the nonessential nature of this enzyme in the unicellular fungus P. pastoris. The ΔFgmt2 mutant exhibited severe growth defects and produced abnormal conidia, while the ΔFgmt1 mutant grew like the wild-type strain, PH-1, under the conditions tested. The ΔFgmt2 mutant also exhibited drastically reduced disease symptoms in wheat and much-delayed disease symptoms in Arabidopsis thaliana. Surprisingly, the ΔFgmt2 mutant was less virulent on different host plants tested than the previously characterized ΔFggcs1 mutant, which lacks GlcCer synthase activity and produces no GlcCer at all. Moreover, the ΔFgmt1 and ΔFgmt2 mutants, as well as the P. pastoris strain in which the C-9-MT gene was deleted, retained sensitivity to the antifungal plant defensins MsDef1 and RsAFP2, indicating that the C-9 methyl group is not a critical structural feature of the GlcCer receptor required for the antifungal action of plant defensins.

scholarly journals Characterization of ASF1 and GCN5 in the ciliated protozoan tetrahymena thermophila

2021 ◽  
Author(s):  
Abdel Rahman Karsou
Keyword(s):  
Tetrahymena Thermophila ◽  
Histone H3 ◽  
Model Organism ◽  
Human Cells ◽  
Ciliated Protozoan ◽  
Yeast Saccharomyces Cerevisiae ◽  
Post Translational Modifications ◽  
Lysine Residues ◽  
Acetyl Group ◽  
H3 Acetylation

One method of regulating accessibility of DNA is chromatin remodelling via histone post-translational modifications (PTM). Adding an acetyl group to the lysine residues (K) on the core histone H3 is one of these chemical modifications. Acetylation of H3 on lysine 56 (H3K56ac) is an important histone alteration that is conserved among most if not all eukaryotes including humans. Several histone acetyl transferases (HAT) have been shown to be responsible for H3K56ac in different organisms including Gen5 and p300/CPB in human cells and Rtt109 in fungi including the yeast Saccharomyces cerevisiae. In addition the histone chaperone ASf1, is also required for these modifications in yeast and human cells. The ciliated protozoan Tetrahymena thermophila is an effective model organism for studying the function of histone PTMs in certain processes including meiosis and RNA interference. Here, I show that tGen5 has H3 acetylation activity and that tAsf1 binds Histone H3.

scholarly journals Characterization of ASF1 and GCN5 in the ciliated protozoan tetrahymena thermophila

2021 ◽  
Author(s):  
Abdel Rahman Karsou
Keyword(s):  
Tetrahymena Thermophila ◽  
Histone H3 ◽  
Model Organism ◽  
Human Cells ◽  
Ciliated Protozoan ◽  
Yeast Saccharomyces Cerevisiae ◽  
Post Translational Modifications ◽  
Lysine Residues ◽  
Acetyl Group ◽  
H3 Acetylation

One method of regulating accessibility of DNA is chromatin remodelling via histone post-translational modifications (PTM). Adding an acetyl group to the lysine residues (K) on the core histone H3 is one of these chemical modifications. Acetylation of H3 on lysine 56 (H3K56ac) is an important histone alteration that is conserved among most if not all eukaryotes including humans. Several histone acetyl transferases (HAT) have been shown to be responsible for H3K56ac in different organisms including Gen5 and p300/CPB in human cells and Rtt109 in fungi including the yeast Saccharomyces cerevisiae. In addition the histone chaperone ASf1, is also required for these modifications in yeast and human cells. The ciliated protozoan Tetrahymena thermophila is an effective model organism for studying the function of histone PTMs in certain processes including meiosis and RNA interference. Here, I show that tGen5 has H3 acetylation activity and that tAsf1 binds Histone H3.

scholarly journals A Novel Sterol Desaturase-Like Protein Promoting Dealkylation of Phytosterols in Tetrahymena thermophila

2011 ◽  
Vol 10 (3) ◽  
pp. 423-434 ◽  
Author(s):  
Mariela L. Tomazic ◽  
Sebastián R. Najle ◽  
Alejandro D. Nusblat ◽  
Antonio D. Uttaro ◽  
Clara B. Nudel
Keyword(s):  
Wild Type Strain ◽  
Tetrahymena Thermophila ◽  
Wild Type ◽  
Knockout Mutant ◽  
Transfer Event ◽  
Content Type ◽  
Ethyl Group ◽  
Fatty Acid Hydroxylase ◽  
Eukaryotic Proteins ◽  
The One

ABSTRACT The gene TTHERM_00438800 ( DES24 ) from the ciliate Tetrahymena thermophila encodes a protein with three conserved histidine clusters, typical of the fatty acid hydroxylase superfamily. Despite its high similarity to sterol desaturase-like enzymes, the phylogenetic analysis groups Des24p in a separate cluster more related to bacterial than to eukaryotic proteins, suggesting a possible horizontal gene transfer event. A somatic knockout of DES24 revealed that the gene encodes a protein, Des24p, which is involved in the dealkylation of phytosterols. Knocked-out mutants were unable to eliminate the C-24 ethyl group from C 29 sterols, whereas the ability to introduce other modifications, such as desaturations at positions C-5(6), C-7(8), and C-22(23), were not altered. Although C-24 dealkylations have been described in other organisms, such as insects, neither the enzymes nor the corresponding genes have been identified to date. Therefore, this is the first identification of a gene involved in sterol dealkylation. Moreover, the knockout mutant and wild-type strain differed significantly in growth and morphology only when cultivated with C 29 sterols; under this culture condition, a change from the typical pear-like shape to a round shape and an alteration in the regulation of tetrahymanol biosynthesis were observed. Sterol analysis upon culture with various substrates and inhibitors indicate that the removal of the C-24 ethyl group in Tetrahymena may proceed by a mechanism different from the one currently known.

scholarly journals Virulence Gene Regulation by the agr System in Clostridium perfringens

2009 ◽  
Vol 191 (12) ◽  
pp. 3919-3927 ◽  
Author(s):  
Kaori Ohtani ◽  
Yonghui Yuan ◽  
Sufi Hassan ◽  
Ruoyu Wang ◽  
Yun Wang ◽  
...  
Keyword(s):  
Mutant Strain ◽  
Clostridium Perfringens ◽  
Culture Supernatant ◽  
Toxin Production ◽  
Toxin Gene ◽  
Wild Type ◽  
Knockout Mutant ◽  
Double Knockout ◽  
Signal Molecule

ABSTRACT A gram-positive anaerobic pathogen, Clostridium perfringens, causes clostridial myonecrosis or gas gangrene in humans by producing numerous extracellular toxins and enzymes that act in concert to degrade host tissue. The agr system is known to be important for the regulation of virulence genes in a quorum-sensing manner in Staphylococcus aureus. A homologue for S. aureus agrBD (agrBDSa ) was identified in the C. perfringens strain 13 genome, and the role of C. perfringens agrBD (agrBDCp ) was examined. The agrBDCp knockout mutant did not express the theta-toxin gene, and transcription of the alpha- and kappa-toxin genes was also significantly decreased in the mutant strain. The mutant strain showed a recovery of toxin production after the addition of the culture supernatant of the wild-type strain, indicating that the agrBDCp mutant lacks a signal molecule in the culture supernatant. An agr-virR double-knockout mutant was constructed to examine the role of the VirR/VirS two-component regulatory system, a key virulence regulator, in agrBDCp -mediated regulation of toxin production. The double-mutant strain could not be stimulated for toxin production with the wild-type culture supernatant. These results indicate that the agrBDCp system plays an important role in virulence regulation and also suggest that VirR/VirS is required for sensing of the extracellular signal and activation of toxin gene transcription in C. perfringens.

Mutational analysis of patterning of oral structures in Tetrahymena

Development ◽  
1984 ◽  
Vol 82 (1) ◽  
pp. 41-66
Author(s):  
Joseph Frankel ◽  
Leslie M. Jenkins ◽  
Julita Bakowska ◽  
E. Marlo Nelsen
Keyword(s):  
Basal Body ◽  
Spatial Organization ◽  
Mutational Analysis ◽  
Tetrahymena Thermophila ◽  
Large Range ◽  
Wild Type ◽  
Ciliated Protozoan ◽  
Constant Length ◽  
Ordered Arrays ◽  
Parallel Arrays

The oral apparatus (OA) of the ciliated protozoan Tetrahymena thermophila consists of four ordered arrays of ciliary units. In wild-type cells, these arrays are constant in spatial organization and vary little in size except during extreme starvation. Recessive mutationsat five gene loci are known to increase the size of the OA. They do this by increasing the lengthof the ciliary arrays, without affecting their width and often without increasing their number beyond the usual four. Comparison of the oral arrays over a large range of sizes has revealed: (1) that the lengths of the anterior two of three parallel arrays (membranelles) are rather tightly coordinated; (2) that the specific basal body configurations resulting from remodelling of the membranelles are only slightly affected by large changes in lengths of membranelles; and (3) that the third membranelle is restricted to a nearly constant length, except in the very largest OAs in which the structure is lengthened but interrupted by a gap in the middle. This gap may reveal thespatial extent of a putative zone of basal body regression. These phenomena are not specific to any of the genotypes utilized in this investigation; the effect of the mutations is to loosen quantitative restrictions and thus reveal underlying associations and constraints.

scholarly journals Phosphorylation-Dependent Targeting of Tetrahymena HP1 to Condensed Chromatin

mSphere ◽  
2016 ◽  
Vol 1 (4) ◽  
Author(s):  
Katerina Yale ◽  
Alan J. Tackett ◽  
Monica Neuman ◽  
Emily Bulley ◽  
Brian T. Chait ◽  
...  
Keyword(s):  
Posttranslational Modifications ◽  
Tetrahymena Thermophila ◽  
Chromatin Condensation ◽  
Histone H3 ◽  
Chromatin Protein ◽  
Ciliated Protozoan ◽  
Cellular Mechanisms ◽  
Content Type ◽  
Evolutionarily Conserved ◽  
Transcription And Replication

ABSTRACT Compacting the genome to various degrees influences processes that use DNA as a template, such as gene transcription and replication. This project was aimed at learning more about the cellular mechanisms that control genome compaction. Posttranslational modifications of proteins involved in genome condensation are emerging as potentially important points of regulation. To help elucidate protein modifications and how they affect the function of condensation proteins, we investigated the phosphorylation of the chromatin protein called Hhp1 in the ciliated protozoan Tetrahymena thermophila. This is one of the first functional investigations of these modifications of a nonhistone chromatin condensation protein that acts on the ciliate genome, and discoveries will aid in identifying common, evolutionarily conserved strategies that control the dynamic compaction of genomes. The evolutionarily conserved proteins related to heterochromatin protein 1 (HP1), originally described in Drosophila, are well known for their roles in heterochromatin assembly and gene silencing. Targeting of HP1 proteins to specific chromatin locales is mediated, at least in part, by the HP1 chromodomain, which binds to histone H3 methylated at lysine 9 that marks condensed regions of the genome. Mechanisms that regulate HP1 targeting are emerging from studies with yeast and metazoans and point to roles for posttranslational modifications. Here, we report that modifications of an HP1 homolog (Hhp1) in the ciliate model Tetrahymena thermophila correlated with the physiological state and with nuclear differentiation events involving the restructuring of chromatin. Results support the model in which Hhp1 chromodomain binds lysine 27-methylated histone H3, and we show that colocalization with this histone mark depends on phosphorylation at a single Cdc2/Cdk1 kinase site in the “hinge region” adjacent to the chromodomain. These findings help elucidate important functional roles of reversible posttranslational modifications of proteins in the HP1 family, in this case, regulating the targeting of a ciliate HP1 to chromatin regions marked with methylated H3 lysine 27. IMPORTANCE Compacting the genome to various degrees influences processes that use DNA as a template, such as gene transcription and replication. This project was aimed at learning more about the cellular mechanisms that control genome compaction. Posttranslational modifications of proteins involved in genome condensation are emerging as potentially important points of regulation. To help elucidate protein modifications and how they affect the function of condensation proteins, we investigated the phosphorylation of the chromatin protein called Hhp1 in the ciliated protozoan Tetrahymena thermophila. This is one of the first functional investigations of these modifications of a nonhistone chromatin condensation protein that acts on the ciliate genome, and discoveries will aid in identifying common, evolutionarily conserved strategies that control the dynamic compaction of genomes.

scholarly journals Loss of Histone H3 K79 Methyltransferase Dot1l Facilitates Kidney Fibrosis by Upregulating Endothelin 1 through Histone Deacetylase 2

2019 ◽  
Vol 31 (2) ◽  
pp. 337-349 ◽  
Author(s):  
Long Zhang ◽  
Lihe Chen ◽  
Chao Gao ◽  
Enuo Chen ◽  
Andrea R. Lightle ◽  
...  
Keyword(s):  
Knockout Mice ◽  
Collecting Duct ◽  
Histone H3 ◽  
Conditional Knockout ◽  
Wild Type ◽  
Double Knockout ◽  
Kidney Fibrosis ◽  
Histone Deacetylase 2 ◽  
Endothelin 1 ◽  
Connecting Tubule

BackgroundThe progression rate of CKD varies substantially among patients. The genetic and epigenetic contributions that modify how individual patients respond to kidney injury are largely unknown. Emerging evidence has suggested that histone H3 K79 methyltransferase Dot1l has an antifibrotic effect by repressing Edn1, which encodes endothelin 1 in the connecting tubule/collecting duct.MethodsTo determine if deletion of the Dot1l gene is a genetic and epigenetic risk factor through regulating Edn1, we studied four groups of mice: wild-type mice, connecting tubule/collecting duct–specific Dot1l conditional knockout mice (Dot1lAC), Dot1l and Edn1 double-knockout mice (DEAC), and Edn1 connecting tubule/collecting duct–specific conditional knockout mice (Edn1AC), under three experimental conditions (streptozotocin-induced diabetes, during normal aging, and after unilateral ureteral obstruction). We used several approaches (colocalization, glutathione S-transferase pulldown, coimmunoprecipitation, yeast two-hybrid, gel shift, and chromatin immunoprecipitation assays) to identify and confirm interaction of Dot1a (the major Dot1l splicing variant in the mouse kidney) with histone deacetylase 2 (HDAC2), as well as the function of the Dot1a-HDAC2 complex in regulating Edn1 transcription.ResultsIn each case, Dot1lAC mice developed more pronounced kidney fibrosis and kidney malfunction compared with wild-type mice. These Dot1lAC phenotypes were ameliorated in the double-knockout DEAC mice. The interaction between Dot1a and HDAC2 prevents the Dot1a-HDAC2 complex from association with DNA, providing a counterbalancing mechanism governing Edn1 transcription by modulating H3 K79 dimethylation and H3 acetylation at the Edn1 promoter.ConclusionsOur study confirms Dot1l to be a genetic and epigenetic modifier of kidney fibrosis, reveals a new mechanism regulating Edn1 transcription by Dot1a and HDAC2, and reinforces endothelin 1 as a therapeutic target of kidney fibrosis.

scholarly journals The contribution of PIP2-type aquaporins to photosynthetic response to increased vapour pressure deficit

2021 ◽  
Author(s):  
D Israel ◽  
S Khan ◽  
C R Warren ◽  
J J Zwiazek ◽  
T M Robson
Keyword(s):  
Vapour Pressure Deficit ◽  
Air Humidity ◽  
Wild Type ◽  
Knockout Mutant ◽  
Evaporative Demand ◽  
Co2 Transport ◽  
Whole Plant ◽  
Knockout Mutants ◽  
Leaf Level ◽  
Low Humidity

Abstract The roles of different plasma membrane aquaporins (PIPs) in leaf-level gas exchange of Arabidopsis thaliana were examined using knockout mutants. Since multiple Arabidopsis PIPs are implicated in CO2 transport across cell membranes, we focused on identifying the effects of the knockout mutations on photosynthesis, and whether they are mediated through the control of stomatal conductance of water vapour (gs), mesophyll conductance of CO2 (gm) or both. We grew Arabidopsis plants in low and high humidity environments and found that the contribution of PIPs to gs was larger under low air humidity when the evaporative demand was high, whereas any effect of lacking PIP function was minimal under higher humidity. The pip2;4 knockout mutant had 44% higher gs than the wild type plants under low humidity, which in turn resulted in an increased net photosynthetic rate (Anet). We also observed a 23% increase in whole-plant transpiration (E) for this knockout mutant. The lack of functional AtPIP2;5 did not affect gs or E, but resulted in homeostasis of gm despite changes of humidity, indicating a possible role in regulating CO2 membrane permeability. CO2 transport measurements in yeast expressing AtPIP2;5 confirmed that this aquaporin is indeed permeable to CO2.

scholarly journals Haploid induction by a maize cenh3 null mutant

2021 ◽  
Vol 7 (4) ◽  
pp. eabe2299 ◽  
Author(s):  
Na Wang ◽  
Jonathan I. Gent ◽  
R. Kelly Dawe
Keyword(s):  
Histone H3 ◽  
Null Mutation ◽  
Null Mutant ◽  
Wild Type ◽  
Haploid Induction ◽  
Cell Divisions ◽  
Gamete Formation ◽  
Simplified Method ◽  
Single Cross ◽  
Subsequent Loss

The production of haploids is an important first step in creating many new plant varieties. One approach used in Arabidopsis involves crossing plants expressing different forms of centromeric histone H3 (CENP-A/CENH3) and subsequent loss of genome with weaker centromeres. However, the method has been ineffective in crop plants. Here, we describe a greatly simplified method based on crossing maize lines that are heterozygous for a cenh3 null mutation. Crossing +/cenh3 to wild-type plants in both directions yielded haploid progeny. Genome elimination was determined by the cenh3 genotype of the gametophyte, suggesting that centromere failure is caused by CENH3 dilution during the postmeiotic cell divisions that precede gamete formation. The cenh3 haploid inducer works as a vigorous hybrid and can be transferred to other lines in a single cross, making it versatile for a variety of applications.

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