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 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.

Mutational analysis of patterning of oral structures in Tetrahymena

Development ◽  
1984 ◽  
Vol 82 (1) ◽  
pp. 67-95
Author(s):  
Joseph Frankel ◽  
E. Marlo Nelsen ◽  
Julita Bakowska ◽  
Leslie M. Jenkins
Keyword(s):  
Mutational Analysis ◽  
Tetrahymena Thermophila ◽  
The State ◽  
The Other ◽  
Basal Bodies ◽  
Ciliated Protozoan ◽  
Vertebrate Limb ◽  
Spatial Configurations ◽  
Oral Development ◽  
Mutant Cells

The ciliary arrays of the oral apparatus of the ciliated protozoan Tetrahymena thermophila each have their own unique ‘pattern signature’, which varies little so long as the number of arrays remains the same. In this study, we analyse the consequence of increases in the number of these arrays (membranelles) brought about by certain mutations. In oral apparatuses of mutant cells, the addition of a membranelle is associated with specific alterations in at least one of the other membranelles. The features that are altered include the relative lengths of membranelles, the state of ciliation of basal bodies located at specific positions within these membranelles, and the spatial configurations resulting from displacement of ciliary units during late oral development. The final organization of each membranelle depends upon its relativeposition along the length of the oral apparatus. This indicates that the membranelles are not individually ‘named’ by the organism, and suggests that the unit of pattern organizationis the membranelle field as a whole. In the Discussion, we consider means for testing whether thesame underlying idea might also apply to multicellular systems, such as the vertebrate limb, in which spatially ordered differences appear to be superimposed upon a fundamental repeating pattern.

scholarly journals Mutational analysis of centrin: an EF-hand protein associated with three distinct contractile fibers in the basal body apparatus of Chlamydomonas.

1992 ◽  
Vol 119 (6) ◽  
pp. 1613-1624 ◽  
Author(s):  
B E Taillon ◽  
S A Adler ◽  
J P Suhan ◽  
J W Jarvik
Keyword(s):  
Amino Acid ◽  
Basal Body ◽  
Calcium Binding ◽  
Mutational Analysis ◽  
Structural Defects ◽  
Wild Type ◽  
Immunofluorescence Analysis ◽  
Ef Hand ◽  
Level 1 ◽  
Ef Hands

Centrin, a 20-kD phosphoprotein with four calcium-binding EF-hands, is present in the centrosome/basal body apparatus of the green alga Chlamydomonas reinhardtii in three distinct locations: the nucleus-basal body connectors, the distal striated fibers, and the flagellar transition regions. In each location, centrin is found in fibrous structures that display calcium-mediated contraction. The mutant vfl2 has structural defects at all of these locations and is defective for basal body localization and/or segregation. We show that the vfl2 mutation is a G-to-A transition in the centrin structural gene which converts a glutamic acid to a lysine at position 101, the first amino acid of the E-helix of the protein's third EF-hand. This proves that centrin is required to construct the nucleus-basal body connectors, the distal striated fibers, and the flagellar transition regions, and it demonstrates the importance of amino acid 101 to normal centrin function. Based on immunofluorescence analysis using anti-centrin antibodies, it appears that vfl2 centrin is capable of binding to the basal body but is incapable of polymerizing into filamentous structures. 19 phenotypic revertants of vfl2 were isolated, and 10 of them, each of which had undergone further mutation at codon 101, were examined in detail. At the DNA level, 1 of the 10 was wild type, and the other 9 were pseudorevertants encoding centrins with the amino acids asparagine, threonine, methionine, or isoleucine at position 101. No ultrastructure defects were apparent in the revertants with asparagine or threonine at position 101, but in those with methionine or isoleucine at position 101, the distal striated fibers were found to be incomplete, indicating that different amino acid substitutions at position 101 can differentially affect the assembly of the three distinct centrin-containing fibrous structures associated with the Chlamydomonas centrosome.

Mutational analysis of regulated exocytosis in Tetrahymena

1998 ◽  
Vol 111 (1) ◽  
pp. 131-140 ◽  
Author(s):  
S.M. Melia ◽  
E.S. Cole ◽  
A.P. Turkewitz
Keyword(s):  
Genetic Analysis ◽  
Mutational Analysis ◽  
Tetrahymena Thermophila ◽  
Differential Centrifugation ◽  
Wild Type ◽  
Granule Formation ◽  
Regulated Exocytosis ◽  
Visual Assay ◽  
Three Stages ◽  
Last Stage

Genetic analysis of regulated exocytosis can be accomplished in ciliates, since mutants defective in stimulus-dependent secretion of dense-core vesicles can be identified. In Tetrahymena thermophila, secretion in wild-type cells can result in their encapsulation by the proteins released from vesicle cores. Cells with defects in secretion were isolated from mutagenized homozygous cells that were generated using a highly efficient method. Screening was based both on a visual assay for encapsulation, and on a novel panning step using differential centrifugation to take advantage of the selective mobility of mutants that fail to encapsulate upon stimulation. 18 mutants with defects in several ordered steps have been identified. Defects in a set of these could be localized to three stages: granule formation, transport to cell surface docking sites, and exocytosis itself. Mutants with defects in this last stage can be ordered into successive steps based on several criteria, including their responsiveness to multiple secretagogues and Ca2+ ionophores. The results of both somatic and genetic complementation on selected pairs also help to characterize the defective factors.

scholarly journals Replication of an rRNA gene origin plasmid in the Tetrahymena thermophila macronucleus is prevented by transcription through the origin from an RNA polymerase I promoter.

1995 ◽  
Vol 15 (6) ◽  
pp. 3372-3381 ◽  
Author(s):  
W J Pan ◽  
R C Gallagher ◽  
E H Blackburn
Keyword(s):  
Rna Polymerase ◽  
Tetrahymena Thermophila ◽  
Rna Polymerase I ◽  
Rrna Gene ◽  
Wild Type ◽  
Independent Evidence ◽  
Origin Region ◽  
Origin Function ◽  
Polymerase I ◽  
Wild Type Promoter

In the somatic macronucleus of the ciliate Tetrahymena thermophila, the palindromic rRNA gene (rDNA) minichromosome is replicated from an origin near the center of the molecule in the 5' nontranscribed spacer. The replication of this rDNA minichromosome is under both cell cycle and copy number control. We addressed the effect on origin function of transcription through this origin region. A construct containing a pair of 1.9-kb tandem direct repeats of the rDNA origin region, containing the origin plus a mutated (+G), but not a wild type, rRNA promoter, is initially maintained in macronuclei as an episome. Late, linear and circular replicons with long arrays of tandem repeats accumulate (W.-J. Pan and E. H. Blackburn, Nucleic Acids Res, in press). We present direct evidence that the +G mutation inactivates this rRNA promoter. It lacks the footprint seen on the wild-type promoter and produces no detectable in vivo transcript. Independent evidence that the failure to maintain wild-type 1.9-kb repeats was caused by transcription through the origin came from placing a short DNA segment containing the rRNA gene transcriptional termination region immediately downstream of the wild-type rRNA promoter. Insertion of this terminator sequence in the correct, but not the inverted, orientation restored plasmid maintenance. Hence, origin function was restored by inactivating the rRNA promoter through the +G mutation or causing termination before transcripts from a wild-type promoter reached the origin region. We propose that transcription by RNA polymerase I through the rDNA origin inhibits replication by preventing replication factors from assembling at the origin.

Structure and expression of two temperature-specific surface proteins in the ciliated protozoan Tetrahymena thermophila

1986 ◽  
Vol 6 (9) ◽  
pp. 3240-3245
Author(s):  
G A Bannon ◽  
R Perkins-Dameron ◽  
A Allen-Nash
Keyword(s):  
Cell Surface ◽  
Specific Surface ◽  
Incubation Temperature ◽  
Tetrahymena Thermophila ◽  
Sodium Dodecyl ◽  
Surface Protein ◽  
Surface Proteins ◽  
Ciliated Protozoan ◽  
Temperature Dependent ◽  
Molecular Sizes

The presence of specific proteins (known as immobilization antigens) on the surface of the ciliated protozoan Tetrahymena thermophila is under environmental regulation. There are five different classes (serotypes) of surface proteins which appear on the cell surface when T. thermophila is cultured under different conditions of temperature or incubation medium; three of these are temperature dependent. The appearance of these proteins on the cell surface is mutually exclusive. We used polyclonal antibodies raised against 30 degrees C (designated SerH3)- and 40 degrees C (designated SerT)-specific surface antigens to study their structure and expression. We showed that these surface proteins contain at least one disulfide bridge. On sodium dodecyl sulfate-denaturing polyacrylamide gels, the nonreduced 30 degrees C- and 40 degrees C-specific surface proteins migrated with molecular sizes of 69 and 36 kilodaltons, respectively. The reduced forms of the proteins migrated with molecular sizes of 58 and 30 kilodaltons, respectively. The synthesis of the surface proteins responded rapidly and with a time course similar to that of the incubation temperature. The synthesis of each surface protein was greatly reduced within 1 h and undetectable by 2 h after a shift to the temperature at which the protein is not expressed. Surface protein synthesis resumed by the end of 1 h after a shift to the temperature at which the protein is expressed. The temperature-dependent induction of these surface proteins appears to be dependent on the synthesis of new mRNA, as indicated by a sensitivity to actinomycin D. Surface protein syntheses were mutually exclusive except at a transition temperature. At 35 degrees C both surface proteins were synthesized by a cell population. These data support the potential of this system as a model for the study of the effects of environmental factors on the genetic regulation of cell surface proteins.

Development of surface pattern during division in Paramecium. II. Defective spatial control in the mutant kin241

Development ◽  
1992 ◽  
Vol 115 (1) ◽  
pp. 319-335 ◽  
Author(s):  
M. Jerka-Dziadosz ◽  
N. Garreau de Loubresse ◽  
J. Beisson
Keyword(s):  
Basal Body ◽  
Major Effect ◽  
The Body ◽  
Cortical Reorganization ◽  
Surface Pattern ◽  
Recessive Mutation ◽  
Wild Type ◽  
Cortical Organization ◽  
Nuclear Reorganization ◽  
In The Wild

kin241 is a monogenic nuclear recessive mutation producing highly pleiotropic effects on cell size and shape, generation time, thermosensitivity, nuclear reorganization and cortical organization. We have analyzed the nature of the cortical disorders and their development during division, using various specific antibodies labelling either one of the cortical cytoskeleton components, as was previously done for analysis of cortical pattern formation in the wild type. Several abnormalities in basal body properties were consistently observed, although with a variable frequency: extra microtubules in either the triplets or in the lumen; nucleation of a second kinetodesmal fiber; abnormal orientation of the newly formed basal body with respect to the mother one. The latter effect seems to account for the major observed cortical disorders (reversal, intercalation of supplementary ciliary rows). The second major effect of the mutation concerns the spatiotemporal map of cortical reorganization during division. Excess basal body proliferation occurs and is correlated with modified boundaries of some of the cortical domains identified in the wild type on the basis of their basal body duplication pattern. This is the first mutant described in a ciliate in which both the structure and duplication of basal bodies and the body plan are affected. The data support the conclusion that the mutation does not alter the nature of the morphogenetic signal(s) which pervade the dividing cell, nor the competence of cytoskeletal structures to respond to signalling, but affects the local interpretation of the signals.

scholarly journals Individual Palmitoyl Residues Serve Distinct Roles in H-Ras Trafficking, Microlocalization, and Signaling

2005 ◽  
Vol 25 (15) ◽  
pp. 6722-6733 ◽  
Author(s):  
Sandrine Roy ◽  
Sarah Plowman ◽  
Barak Rotblat ◽  
Ian A. Prior ◽  
Cornelia Muncke ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Golgi Apparatus ◽  
Lipid Rafts ◽  
Lipid Bilayer ◽  
Spatial Organization ◽  
Cysteine Residues ◽  
Wild Type ◽  
Membrane Affinity ◽  
Lateral Segregation

ABSTRACT H-ras is anchored to the plasma membrane by two palmitoylated cysteine residues, Cys181 and Cys184, operating in concert with a C-terminal S-farnesyl cysteine carboxymethylester. Here we demonstrate that the two palmitates serve distinct biological roles. Monopalmitoylation of Cys181 is required and sufficient for efficient trafficking of H-ras to the plasma membrane, whereas monopalmitoylation of Cys184 does not permit efficient trafficking beyond the Golgi apparatus. However, once at the plasma membrane, monopalmitoylation of Cys184 supports correct GTP-regulated lateral segregation of H-ras between cholesterol-dependent and cholesterol-independent microdomains. In contrast, monopalmitoylation of Cys181 dramatically reverses H-ras lateral segregation, driving GTP-loaded H-ras into cholesterol-dependent microdomains. Intriguingly, the Cys181 monopalmitoylated H-ras anchor emulates the GTP-regulated microdomain interactions of N-ras. These results identify N-ras as the Ras isoform that normally signals from lipid rafts but also reveal that spacing between palmitate and prenyl groups influences anchor interactions with the lipid bilayer. This concept is further supported by the different plasma membrane affinities of the monopalmitoylated anchors: Cys181-palmitate is equivalent to the dually palmitoylated wild-type anchor, whereas Cys184-palmitate is weaker. Thus, membrane affinity of a palmitoylated anchor is a function both of the hydrophobicity of the lipid moieties and their spatial organization. Finally we show that the plasma membrane affinity of monopalmitoylated anchors is absolutely dependent on cholesterol, identifying a new role for cholesterol in promoting interactions with the raft and nonraft plasma membrane.

scholarly journals A method for mapping germ line sequences in Tetrahymena thermophila using the polymerase chain reaction.

Genetics ◽  
1994 ◽  
Vol 137 (1) ◽  
pp. 95-106 ◽  
Author(s):  
D Cassidy-Hanley ◽  
M C Yao ◽  
P J Bruns
Keyword(s):  
Polymerase Chain Reaction ◽  
Dna Sequences ◽  
Tetrahymena Thermophila ◽  
Germ Line ◽  
Repetitive Sequences ◽  
Mapping Technique ◽  
Ciliated Protozoan ◽  
Chain Reaction ◽  
Polymerase Chain ◽  
Template Dna

Abstract A method for mapping DNA sequences to specific germinal chromosomes in the ciliated protozoan Tetrahymena thermophila has been developed. This mapping technique (PCR mapping) utilizes the polymerase chain reaction and template DNA derived from nullisomic strains to directly assign micronuclear DNA sequences to specific micronuclear chromosomes. Using this technique, a number of unique sequences and short repetitive sequences flanked by unique sequences have been mapped to four of the five germinal chromosomes.

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

1986 ◽  
Vol 6 (7) ◽  
pp. 2364-2370 ◽  
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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