scholarly journals ε-Tubulin Is an Essential Component of the Centriole

2002 ◽  
Vol 13 (11) ◽  
pp. 3859-3869 ◽  
Author(s):  
Susan K. Dutcher ◽  
Naomi S. Morrissette ◽  
Andrea M. Preble ◽  
Craig Rackley ◽  
John Stanga
Keyword(s):  
Basal Body ◽  
Positional Cloning ◽  
Stop Codon ◽  
Polyclonal Antibodies ◽  
Premature Stop Codon ◽  
Full Length ◽  
Basal Bodies ◽  
Cleavage Furrow ◽  
Essential Component ◽  
Flagellar Assembly

Centrioles and basal bodies are cylinders composed of nine triplet microtubule blades that play essential roles in the centrosome and in flagellar assembly. Chlamydomonas cells with thebld2-1 mutation fail to assemble doublet and triplet microtubules and have defects in cleavage furrow placement and meiosis. Using positional cloning, we have walked 720 kb and identified a 13.2-kb fragment that contains ε-tubulin and rescues the Bld2 defects. The bld2-1 allele has a premature stop codon and intragenic revertants replace the stop codon with glutamine, glutamate, or lysine. Polyclonal antibodies to ε-tubulin show peripheral labeling of full-length basal bodies and centrioles. Thus, ε-tubulin is encoded by the BLD2 allele and ε-tubulin plays a role in basal body/centriole morphogenesis.

Extragenic Bypass Suppressors of Mutations in the Essential Gene BLD2 Promote Assembly of Basal Bodies With Abnormal Microtubules in Chlamydomonas reinhardtii

Genetics ◽  
2001 ◽  
Vol 157 (1) ◽  
pp. 163-181
Author(s):  
Andrea M Preble ◽  
Thomas H Giddings ◽  
Susan K Dutcher
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Basal Body ◽  
Insertional Mutagenesis ◽  
Essential Gene ◽  
Basal Bodies ◽  
Cleavage Furrow ◽  
Wild Type ◽  
Lethal Phenotype ◽  
Flagellar Assembly ◽  
And Function

Abstract bld2-1 mutant Chlamydomonas reinhardtii strains assemble basal bodies with singlet microtubules; bld2-1 cells display flagellar assembly defects as well as positioning defects of the mitotic spindle and cleavage furrow. To further understand the role of the BLD2 gene, we have isolated three new bld2 alleles and three partially dominant extragenic suppressors, rgn1-1, rgn1-2, and rgn1-3. bld2 rgn1-1 strains have phenotypes intermediate between those of bld2 and wild-type strains with respect to flagellar number, microtubule rootlet organization, cleavage furrow positioning, and basal body structural phenotypes. Instead of the triplet microtubules of wild-type cells, bld2 rgn1-1 basal bodies have mixtures of no, singlet, doublet, and triplet microtubules. The bld2-4 allele was made by insertional mutagenesis and identified in a noncomplementation screen in a diploid strain. The bld2-4 allele has a lethal phenotype based on mitotic segregation in diploid strains and in haploid strains generated by meiotic recombination. The lethal phenotype in haploid strains is suppressed by rgn1-1; these suppressed strains have similar phenotypes to other bld2 rgn1-1 double mutants. It is likely that BLD2 is an essential gene that is needed for basal body assembly and function.

scholarly journals The ultrastructure of cell division in Euglena gracilis

1978 ◽  
Vol 31 (1) ◽  
pp. 25-35
Author(s):  
M.A. Gillott ◽  
R.E. Triemer
Keyword(s):  
Cell Division ◽  
Nuclear Envelope ◽  
Basal Body ◽  
Euglena Gracilis ◽  
Equatorial Region ◽  
Basal Bodies ◽  
Cleavage Furrow ◽  
Free Zone ◽  
Prophase Nucleus

The ultrastructure of mitosis in Euglena gracilis was investigated. At preprophase the nucleus migrates anteriorly and associates with the basal bodies. Flagella and basal bodies replicate at preprophase. Cells retain motility throughout division. The reservoir and the prophase nucleus elongate perpendicular to the incipient cleavage furrow. One basal body pair surrounded by a ribosome-free zone is found at each of the nuclear poles. The spindle forms within the intact nuclear envelope- Polar fenestrae are absent. At metaphase, the endosome is elongated from pole to pole, and chromosomes are loosely arranged in the equatorial region. Distinct, trilayered kinetochores are present. Spindle elongates as chromosomes migrate to the poles forming a dumb-bell shaped nucleus by telophase. Daughter nuclei are formed by constriction of the nuclear envelope. Cytokinesis is accomplished by furrowing. Cell division in Euglena is compared with that of certain other algae.

scholarly journals Synergistic Activation of Defense Responses in Arabidopsis by Simultaneous Loss of the GSL5 Callose Synthase and the EDR1 Protein Kinase

2010 ◽  
Vol 23 (5) ◽  
pp. 578-584 ◽  
Author(s):  
Anna Wawrzynska ◽  
Natalie L. Rodibaugh ◽  
Roger W. Innes
Keyword(s):  
Powdery Mildew ◽  
Positional Cloning ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Defense Responses ◽  
Loss Of Function ◽  
Callose Synthase ◽  
Synergistic Activation ◽  
Golovinomyces Cichoracearum ◽  
Inducible Gene

Loss-of-function mutations in the EDR1 gene of Arabidopsis confer enhanced resistance to Golovinomyces cichoracearum (powdery mildew). Disease resistance mediated by the edr1 mutation is dependent on an intact salicylic acid (SA) signaling pathway, but edr1 mutant plants do not constitutively express the SA-inducible gene PR-1 and are not dwarfed. To identify other components of the EDR1 signaling network, we screened for mutations that enhanced the edr1 mutant phenotype. Here, we describe an enhancer of edr1 mutant, eed3, which forms spontaneous lesions in the absence of pathogen infection, constitutively expresses both SA- and methyl jasmonate (JA)–inducible defense genes, and is dwarfed. Positional cloning of eed3 revealed that the mutation causes a premature stop codon in GLUCAN SYNTHASE-LIKE 5 (GSL5, also known as POWDERY MILDEW RESISTANT 4), which encodes a callose synthase required for pathogen-induced callose production. Significantly, gsl5 single mutants do not constitutively express PR-1 or AtERF1 (a JA-inducible gene) and are not dwarfed. Thus, loss of both EDR1 and GSL5 function has a synergistic effect. Our data suggest that EDR1 and GSL5 negatively regulate SA and JA production or signaling by independent mechanisms and that negative regulation of defense signaling by GSL5 may be independent of callose production.

scholarly journals YPK9 and WHI2 Negatively Interact during Oxidative Stress

2021 ◽  
Vol 9 (12) ◽  
pp. 2584
Author(s):  
Florenal Joseph ◽  
Darach Miller ◽  
Oleg V. Evgrafov ◽  
William J. Chirico
Keyword(s):  
Oxidative Stress ◽  
Hydrogen Peroxide ◽  
Genetic Interaction ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Full Length ◽  
Deletion Strain ◽  
Acid Protein ◽  
Insight Into ◽  
Amino Acid Protein

Yeast PARK9 (YPK9) shares homology with human ATP13A2, which encodes a polyamine transporter implicated in juvenile forms of Parkinson’s disease. We used YPK9 to gain insight into how ATP13A2 affects cell growth and sensitivity to oxidative stress. Surprisingly, the YPK9 deletion strain from the Saccharomyces cerevisiae deletion collection (YKO) in wildtype BY4741 (mating type a) grew faster and was more resistant to hydrogen peroxide than a commercial, putative parental BY4741 wildtype strain (BY4741COM). In contrast, deleting YPK9 from BY4741COM rendered it very sensitive to hydrogen peroxide, suggesting its background is different from that of the deletion collection. Whole-genome sequencing revealed that BY4741COM and BY4741COMypk9∆ contain a novel premature stop codon near the 3′ end of WHI2 (WHI2G1324T), whereas the collection’s YPK9 deletion strain contains WHI2, which encodes a 486 amino acid protein, Whi2p. Replacing full-length WHI2 with the sequence coding for the predicted truncation (Whi2pE442*) rendered strains more sensitive to hydrogen peroxide, whereas the converse replacement rendered them more resistant. The sequences of WHI2 in 20 randomly chosen strains from the collection encode the full-length protein, indicating that the putative parental BY4741 WHI2G1324T strain’s genetic background differs from that of the deletion collection. Examination of WHI2 sequences in several commonly used wildtype S. cerevisiae strains and isolates revealed other Whi2p truncations that might yield altered phenotypes. Together, these results demonstrate a novel premature stop codon in WHI2 that renders yeast sensitive to hydrogen peroxide; they also reveal a negative genetic interaction between WHI2 and YPK9 in the presence of hydrogen peroxide in the BY4741 background.

scholarly journals Patient-specific iPSC-derived photoreceptor precursor cells as a means to investigate retinitis pigmentosa

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Budd A Tucker ◽  
Robert F Mullins ◽  
Luan M Streb ◽  
Kristin Anfinson ◽  
Mari E Eyestone ◽  
...  
Keyword(s):  
Pluripotent Stem Cells ◽  
Protein Misfolding ◽  
Autosomal Recessive ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Photoreceptor Cells ◽  
Precursor Cells ◽  
Patient Specific ◽  
Basal Bodies ◽  
Induced Pluripotent

Next-generation and Sanger sequencing were combined to identify disease-causing USH2A mutations in an adult patient with autosomal recessive RP. Induced pluripotent stem cells (iPSCs), generated from the patient’s keratinocytes, were differentiated into multi-layer eyecup-like structures with features of human retinal precursor cells. The inner layer of the eyecups contained photoreceptor precursor cells that expressed photoreceptor markers and exhibited axonemes and basal bodies characteristic of outer segments. Analysis of the USH2A transcripts of these cells revealed that one of the patient’s mutations causes exonification of intron 40, a translation frameshift and a premature stop codon. Western blotting revealed upregulation of GRP78 and GRP94, suggesting that the patient’s other USH2A variant (Arg4192His) causes disease through protein misfolding and ER stress. Transplantation into 4-day-old immunodeficient Crb1−/− mice resulted in the formation of morphologically and immunohistochemically recognizable photoreceptor cells, suggesting that the mutations in this patient act via post-developmental photoreceptor degeneration.

scholarly journals The Uni2 Phosphoprotein is a Cell Cycle–regulated Component of the Basal Body Maturation Pathway in Chlamydomonas reinhardtii

2008 ◽  
Vol 19 (1) ◽  
pp. 262-273 ◽  
Author(s):  
Brian P. Piasecki ◽  
Matthew LaVoie ◽  
Lai-Wa Tam ◽  
Paul A. Lefebvre ◽  
Carolyn D. Silflow
Keyword(s):  
Cell Cycle ◽  
Chlamydomonas Reinhardtii ◽  
Basal Body ◽  
Basal Bodies ◽  
Mitotic Progression ◽  
Transition Zones ◽  
Phosphatase Treatment ◽  
Flagellar Assembly ◽  
A Cell ◽  
Sequential Assembly

Mutations in the UNI2 locus in Chlamydomonas reinhardtii result in a “uniflagellar” phenotype in which flagellar assembly occurs preferentially from the older basal body and ultrastructural defects reside in the transition zones. The UNI2 gene encodes a protein of 134 kDa that shares 20.5% homology with a human protein. Immunofluorescence microscopy localized the protein on both basal bodies and probasal bodies. The protein is present as at least two molecular-weight variants that can be converted to a single form with phosphatase treatment. Synthesis of Uni2 protein is induced during cell division cycles; accumulation of the phosphorylated form coincides with assembly of transition zones and flagella at the end of the division cycle. Using the Uni2 protein as a cell cycle marker of basal bodies, we observed migration of basal bodies before flagellar resorption in some cells, indicating that flagellar resorption is not required for mitotic progression. We observed the sequential assembly of new probasal bodies beginning at prophase. The uni2 mutants may be defective in the pathways leading to flagellar assembly and to basal body maturation.

scholarly journals Variation inListeria monocytogenesDose Responses in Relation to Subtypes Encoding a Full-Length or Truncated Internalin A

2010 ◽  
Vol 77 (4) ◽  
pp. 1171-1180 ◽  
Author(s):  
Yuhuan Chen ◽  
William H. Ross ◽  
Richard C. Whiting ◽  
Anna Van Stelten ◽  
Kendra K. Nightingale ◽  
...  
Keyword(s):  
Stop Codon ◽  
Premature Stop Codon ◽  
Intestinal Barrier ◽  
Full Length ◽  
Quantitative Evidence ◽  
Consumption Data ◽  
Genes Encoding ◽  
A Cell ◽  
Dose Responses ◽  
Internalin A

ABSTRACTInternalin A (InlA; encoded byinlA) facilitates the crossing of the intestinal barrier byListeria monocytogenes. Mutations leading to a premature stop codon (PMSC) ininlAand thus attenuated mammalian virulence have been reported. We recently characterized 502L. monocytogenesfood isolates from a retail survey and 507 human clinical isolates from multiple U.S. states with respect to the presence/absence ofinlAmutations. The objective of this study was to investigate the hypothesis that dose responses for human listeriosis vary betweenL. monocytogenesstrains with and those without a PMSC ininlA. Subtype-specific prevalence and concentration distributions in food, along with epidemiologic and consumption data, were input into established dose-response models to generate anrvalue (probability of a cell causing illness). Under the conservative assumption thatL. monocytogeneslevels at retail represent levels consumed, mean log10rvalues were −8.1 and −10.7 forL. monocytogenessubtypes with genes encoding a full-length and a truncated InlA, respectively.L. monocytogenescarrying a 5′ frameshift mutation in a homopolymeric tract showed a mean log10rvalue of −12.1. Confidence intervals for thervalues and their differences varied depending on subtypes. When the increase in concentration ofL. monocytogenessubtypes between retail and consumption was considered, mean log10rvalues were reduced to −10.4, −13.8, and −12.8 for the subtypes with genes encoding a full-length InlA, for the subtypes carrying a PMSC ininlA, and for allL. monocytogenesisolates regardless of subtype, respectively. Our study provides further quantitative evidence thatL. monocytogenessubtypes vary in abilities and relative likelihoods of causing human disease, which were mechanistically related to defined genetic markers.

scholarly journals Detection of ALDH3B2 in Human Placenta

2019 ◽  
Vol 20 (24) ◽  
pp. 6292
Author(s):  
Sylwia Michorowska ◽  
Joanna Giebułtowicz ◽  
Renata Wolinowska ◽  
Anna Konopka ◽  
Anna Wilkaniec ◽  
...  
Keyword(s):  
Western Blot ◽  
Human Placenta ◽  
Stop Codon ◽  
Premature Stop Codon ◽  
Premature Termination Codon ◽  
Full Length ◽  
Start Codon ◽  
Phosphorylation Sites ◽  
Putative Open Reading Frame ◽  
Healthy Human

Aldehyde dehydrogenase 3B2 (ALDH3B2) gene contains a premature termination codon, which can be skipped or suppressed resulting in full-length protein expression. Alternatively, the longest putative open reading frame starting with the second in-frame start codon would encode short isoform. No unequivocal evidence of ALDH3B2 expression in healthy human tissues is available. The aim of this study was to confirm its expression in human placenta characterized by the highest ALDH3B2 mRNA abundance. ALDH3B2 DNA and mRNA were sequenced. The expression was investigated using western blot. The identity of the protein was confirmed using mass spectrometry (MS). The predicted tertiary and quaternary structures, subcellular localization, and phosphorylation sites were assessed using bioinformatic analyses. All DNA and mRNA isolates contained the premature stop codon. In western blot analyses, bands corresponding to the mass of full-length protein were detected. MS analysis led to the identification of two unique peptides, one of which is encoded by the nucleotide sequence located upstream the second start codon. Bioinformatic analyses suggest cytoplasmic localization and several phosphorylation sites. Despite premature stop codon in DNA and mRNA sequences, full-length ALDH3B2 was found. It can be formed as a result of premature stop codon readthrough, complex phenomenon enabling stop codon circumvention.

scholarly journals TheUNI3Gene Is Required for Assembly of Basal Bodies ofChlamydomonasand Encodes δ-Tubulin, a New Member of the Tubulin Superfamily

1998 ◽  
Vol 9 (6) ◽  
pp. 1293-1308 ◽  
Author(s):  
Susan K. Dutcher ◽  
Emanuel C. Trabuco
Keyword(s):  
Electron Microscopy ◽  
Cell Cycle ◽  
Basal Body ◽  
Pedigree Analysis ◽  
Basal Bodies ◽  
Cleavage Furrow ◽  
Sequence Identity ◽  
Cycle Dependent ◽  
Single Flagellum

We have cloned the UNI3 gene inChlamydomonas and find that it encodes a new member of the tubulin superfamily. Although Uni3p shares significant sequence identity with α-, β-, and γ-tubulins, there is a region of Uni3p that has no similarity to tubulins or other known proteins. Mutantuni3–1 cells assemble zero, one, or two flagella. Pedigree analysis suggests that flagellar number inuni3–1 cells is a function of the age of the cell. The uniflagellate uni3–1 cells show a positional phenotype; the basal body opposite the eyespot templates the single flagellum. A percentage of uni3–1 cells also fail to orient the cleavage furrow properly, and basal bodies have been implicated in the placement of cleavage furrows in Chlamydomonas. Finally when uni3–1 cells are observed by electron microscopy, doublet rather than triplet microtubules are observed at the proximal end of the basal bodies. We propose that the Uni3 tubulin is involved in both the function and cell cycle-dependent maturation of basal bodies/centrioles.

scholarly journals Cep97 Is Required For Centriole Structural Integrity And Cilia Formation In Drosophila

2019 ◽  
Author(s):  
Jeroen Dobbelaere ◽  
Marketa Schmidt-Cernohorska ◽  
Martina Huranova ◽  
Dea Slade ◽  
Alexander Dammermann
Keyword(s):  
Drosophila Melanogaster ◽  
Basal Body ◽  
Structural Integrity ◽  
Cultured Cells ◽  
Full Length ◽  
Proper Function ◽  
Basal Bodies ◽  
Cell Divisions ◽  
Cilia Formation

SUMMARYCentrioles are highly elaborate microtubule-based structures responsible for the formation of centrosomes and cilia. Despite considerable variation across species and tissues, within any given tissue their size is essentially constant [1, 2]. While the diameter of the centriole cylinder is set by the dimensions of the inner scaffolding structure of the cartwheel [3], how centriole length is set so precisely and stably maintained over many cell divisions is not well understood. Cep97 and CP110 are conserved proteins that localize to the distal end of centrioles and have been reported to limit centriole elongation in vertebrates [4, 5]. Here, we examine Cep97 function in Drosophila melanogaster. We show that Cep97 is essential for formation of full-length centrioles in multiple tissues of the fly. We further identify the microtubule deacetylase Sirt2 as a Cep97 proximity interactor. Deletion of Sirt2 likewise affects centriole size. Interestingly, so does deletion of the acetylase Atat1, indicating that loss of stabilizing acetyl marks impairs centriole integrity. Cep97 and CP110 were originally identified as inhibitors of cilia formation in vertebrate cultured cells [6] and loss of CP110 is a widely used marker of basal body maturation. In contrast, in Drosophila Cep97 is only transiently removed from basal bodies and loss of Cep97 strongly impairs ciliogenesis. Collectively, our results support a model whereby Cep97 functions as part of a protective cap that acts together with the microtubule acetylation machinery to maintain centriole stability, essential for proper function in cilium biogenesis.

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