Dissociation and reassociation of trichocyst proteins: biochemical and ultrastructural studies

1987 ◽  
Vol 87 (1) ◽  
pp. 3-25
Author(s):  
J.B. Peterson ◽  
J.E. Heuser ◽  
D.L. Nelson
Keyword(s):  
Protein Concentration ◽  
Morphological Changes ◽  
Protein Composition ◽  
Reducing Agent ◽  
Paramecium Tetraurelia ◽  
Wild Type ◽  
Ultrastructural Studies ◽  
Cylindrical Shaft ◽  
Experimental Protocols ◽  
Acidic Proteins

Trichocysts, the crystalline exocytotic organelles in Paramecium tetraurelia, are composed of small, acidic proteins existing primarily as disulphide-linked dimers. We have disaggregated trichocyst proteins with heat, simultaneously observing the changes in morphology and protein composition. The tip matrix was most heat-labile; its subunits progressively broke away from the distal end. During this process, breakdown of the cylindrical shaft began. Shafts first became flattened and torn lengthwise, yielding smaller, interconnected pieces still having the crystalline arrangement of their 5 nm thick fibres. Ultimately this pattern became disordered, and discrete fibrils of the same thickness disengaged from the meshwork. In freeze-etched preparations these fibrils were composed of thinner filaments in side-by-side association. Disaggregation of the tip sheath began from the distal end before shaft dissociation was complete. Trichocysts broke down to thin fibrils, but probably not to monomeric subunits. At least three proteins were preferentially released in the initial phase of dissociation. Disulphide-reducing agent present during heating increased the rate of dissociation without altering the sequence of morphological changes or the order of release of individual proteins. The rate and extent of heat-induced dissociation were strongly dependent on pH and cation concentration. The stabilizing effects of low pH and of cations were additive. A cooled suspension of fully dissociated trichocysts reassociated into sedimentable aggregates with discernible filamentous order, but without the crystalline structure of intact trichocysts. Reassociation was dependent upon time, temperature and protein concentration. All but one of the trichocyst proteins re-entered the sedimentable aggregate during reassociation. Reassociation was faster and more complete at pH 6 than at pH 8 and was stimulated by Ca2+, Mg2+ and La3+. Trichocyst proteins dissociated in the presence of dithiothreitol did not reassociate, even after removal of the reducing agent. Trichocysts from mutants defective to varying degrees in trichocyst formation were subjected to similar experimental protocols. Heat-dissociated trichocysts of the mutants scc6 and ptA1 reassociated at rates similar to those of wild-type; ftA3 showed slower reassociation, and tam38 showed little or no reassociation. Reassociation of wild-type trichocyst proteins was blocked by the addition of an equal amount of tam38 trichocyst proteins.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Biochemical studies of the excitable membrane of Paramecium tetraurelia. III. Proteins of cilia and ciliary membranes.

1980 ◽  
Vol 84 (3) ◽  
pp. 717-738 ◽  
Author(s):  
A Adoutte ◽  
R Ramanathan ◽  
R M Lewis ◽  
R R Dute ◽  
K Y Ling ◽  
...  
Keyword(s):  
Membrane Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Protein Composition ◽  
Membrane Vesicles ◽  
Two Dimensions ◽  
Exponential Growth Phase ◽  
Paramecium Tetraurelia ◽  
Wild Type ◽  
Protein Patterns ◽  
Ciliary Membrane

As a first step in the biochemical analysis of membrane excitation in wild-type Paramecium and its behavioral mutants we have defined the protein composition of the ciliary membrane of wild-type cells. The techniques for the isolation of cilia and ciliary membrane vesicles were refined. Membranes of high purity and integrity were obtained without the use of detergents. The fractions were characterized by electron microscopy, and the proteins of whole cilia, axonemes, and ciliary membrane vesicles were resolved by SDS polyacrylamide gel electrophoresis and isoelectric focusing in one and two dimensions. Protein patterns and EM appearance of the fractions were highly reproducible. Over 200 polypeptides were present in isolated cilia, most of which were recovered in the axonemal fraction. Trichocysts, which were sometimes present as a minor contaminant in ciliary preparations, were composed of a very distinct set of over 30 polypeptides of mol wt 11,000--19,000. Membrane vesicles contained up to 70 polypeptides of mol wt 15,000--250,000. The major vesicle species were a high molecular weight protein (the "immobilization antigen") and a group of acidic proteins with mol wt similar to or approximately 40,000. These and several other membrane proteins were specifically decreased or totally absent in the axoneme fraction. Tubulin, the major axonemal species, occurred only in trace amounts in isolated vesicles; the same was true for Tetrahymena ciliary membranes prepared by the methods described in this paper. A protein of mol wt 31,000, pI 6.8, was virtually absent in vesicles prepared from cells in exponential growth phase, but became prominent early in stationary phase in good correlation with cellular mating reactivity. This detailed characterization will provide the basis for comparison of the ciliary proteins of wild-type and behavioral mutants and for analysis of topography and function of membrane proteins. It will also be useful in future studies of trichocysts and mating reactions.

scholarly journals The Cloning and Molecular Analysis of pawn-B in Paramecium tetraurelia

Genetics ◽  
2000 ◽  
Vol 155 (3) ◽  
pp. 1105-1117 ◽  
Author(s):  
W John Haynes ◽  
Kit-Yin Ling ◽  
Robin R Preston ◽  
Yoshiro Saimi ◽  
Ching Kung
Keyword(s):  
Genomic Library ◽  
Open Reading Frame ◽  
Paramecium Tetraurelia ◽  
Wild Type ◽  
Rt Pcr ◽  
Reading Frame ◽  
Close Proximity ◽  
In The Wild ◽  
Dna Fragment ◽  
Alternative Sites

Abstract Pawn mutants of Paramecium tetraurelia lack a depolarization-activated Ca2+ current and do not swim backward. Using the method of microinjection and sorting a genomic library, we have cloned a DNA fragment that complements pawn-B (pwB/pwB). The minimal complementing fragment is a 798-bp open reading frame (ORF) that restores the Ca2+ current and the backward swimming when expressed. This ORF contains a 29-bp intron and is transcribed and translated. The translated product has two putative transmembrane domains but no clear matches in current databases. Mutations in the available pwB alleles were found within this ORF. The d4-95 and d4-96 alleles are single base substitutions, while d4-662 (previously pawn-D) harbors a 44-bp insertion that matches an internal eliminated sequence (IES) found in the wild-type germline DNA except for a single C-to-T transition. Northern hybridizations and RT-PCR indicate that d4-662 transcripts are rapidly degraded or not produced. A second 155-bp IES in the wild-type germline ORF excises at two alternative sites spanning three asparagine codons. The pwB ORF appears to be separated from a 5′ neighboring ORF by only 36 bp. The close proximity of the two ORFs and the location of the pwB protein as indicated by GFP-fusion constructs are discussed.

scholarly journals Phenotypic and Genetic Analysis of “Chameleon,” a Paramecium Mutant With an Enhanced Sensitivity to Magnesium

Genetics ◽  
1997 ◽  
Vol 146 (3) ◽  
pp. 871-880
Author(s):  
Robin R Preston ◽  
Jocelyn A Hammond
Keyword(s):  
Genetic Analysis ◽  
Single Locus ◽  
Behavioral Analysis ◽  
Paramecium Tetraurelia ◽  
Wild Type ◽  
Ion Conductance ◽  
Membrane Excitation ◽  
Enhanced Sensitivity ◽  
Electrophysiological Analysis ◽  
Mutant Strains

Three mutant strains of Paramecium tetraurelia with an enhanced sensitivity to magnesium have been isolated. These new “Chameleon” mutants result from partial- or codominant mutations at a single locus, Cha. Whereas the wild type responded to 5 mm Mg2+ by swimming backward for 10–15 sec, Cha mutants responded with ∼30 sec backward swimming. Electrophysiological analysis suggested that this behavior may be caused by slowing in the rate at which a Mg2+-specific ion conductance deactivates following membrane excitation. This would be consistent with an observed increase in the sensitivity of Cha mutants to nickel poisoning, since Ni2+ is also able to enter the cell via this pathway. More extensive behavioral analysis showed that Cha cells also overresponded to Na+, but there was no evidence for a defect in intracellular Ca2+ homeostasis that might account for a simultaneous enhancement of both the Mg2+ and Na+ conductances. The possibility that the Cha locus may encode a specific regulator of the Mg2+- and Na+-permeabilities is considered.

scholarly journals GENE INTERACTIONS BETWEEN POTASSIUM-SENSITIVE AND POTASSIUM-RESISTANT MUTATIONS OF PARAMECIUM TETRAURELIA

Genetics ◽  
1983 ◽  
Vol 103 (2) ◽  
pp. 153-160
Author(s):  
Donald L Cronkite
Keyword(s):  
High Resistance ◽  
Gene Interactions ◽  
Paramecium Tetraurelia ◽  
Wild Type ◽  
Recessive Mutations ◽  
Resistant Genes ◽  
Type Gene ◽  
Moderately Resistant

ABSTRACT Two unlinked recessive mutations (ks-1 and ks-2) have been induced in Paramecium tetraurelia stock 51. Wild-type survives and grows when up to 30 mm KCl is added to the medium, but the mutants cease to grow and die when added KCl reaches 20-25 m m. These K+-sensitives have been crossed to stocks containing the K+-resistant genes, fA (very resistant) and kA(moderately resistant). All four genes are unlinked. Double mutants of ks-1 and either kA or fA are as resistant as the resistant member of the pair. Doubles of ks-2 and kA are like wild type, and doubles of ks-2 and fA are shifted from high resistance toward wild type. Gene ks-2 acts like a suppressor of kA and fA. This suppression can be understood in terms of the known biochemical defects of the mutants.

scholarly journals Obtaining human hair keratin-based films and their characteristics

2021 ◽  
Vol 15 (1) ◽  
pp. 27-36
Author(s):  
V. V. Mykhaliuk ◽  
◽  
V. V. Havryliak ◽  
Keyword(s):  
Molecular Weight ◽  
Disulfide Bonds ◽  
Protein Concentration ◽  
Sodium Dodecyl ◽  
Protein Composition ◽  
Surface Characteristics ◽  
Film Structure ◽  
X Ray ◽  
Wide Range ◽  
Scanning Electron

Background. Keratins are natural biopolymers with a wide range of applications in the field of biotechnology. Materials and Methods. Extraction of keratins was performed by a modified Nakamura method using 250 mM DTT. The protein concentration in the supernatant was determined by Bradford method. The protein composition was studied by their electro­phoretic separation in a polyacrylamide gel in the presence of sodium dodecyl sulfate. The films were made by casting. The surface characteristics of the films were determined using a scanning electron microscope REMMA-102. The elemental composition of the films was determined using an X-ray microanalyzer. Results. The protein concentration in the supernatant was 3.75 mg/mL. After using dithiothreitol in the extraction mixture, we obtained proteins of intermediate filaments with a molecular weight of 40–60 kDa and a low Sulfur content. In the low molecular weight region, we obtained keratin-associated proteins with a molecular weight of 10–30 kDa and a high content of Sulfur. These proteins belong to fibrillar proteins, which can be used as a matrix for the creation of new keratin-containing biocomposites with a wide range of applications in reparative medicine and tissue engineering. Based on the obtained keratin extract, polymer films with and without the addition of glycerol were made. Scanning electron microscopy revealed that glycerol provided the film structure with homogeneity and plasticity due to the accumulation of moisture after the fixation by water vapor. The X-ray microanalysis of films revealed such elements as Sodium, Silicon, Sulfur, Potassium. Among the detected elements, Sulfur has the largest share that is due to the large number of disulfide bonds in the keratin molecule. Conclusions. The polymer keratin films with the addition of glycerol demonstrated better mechanical properties and can be used in biomedicine.

scholarly journals Identification of DNA segments capable of rescuing a non-mendelian mutant in paramecium.

Genetics ◽  
1994 ◽  
Vol 136 (4) ◽  
pp. 1325-1328
Author(s):  
C S Kim ◽  
J R Preer ◽  
B Polisky
Keyword(s):  
Type A ◽  
Paramecium Tetraurelia ◽  
Wild Type ◽  
Repeat Units

Abstract The non-Mendelian mutant d48 of Paramecium tetraurelia contains micronuclear wild type A genes, but at autogamy and conjugation proper processing fails and new macronuclei lack A genes. When cloned A genes are injected into the macronucleus of d48, proper processing is restored at the next autogamy; d48 is rescued, becoming permanently wild type. In the present study we have injected portions of the A gene into d48. We find that the ability to rescue extends over a large portion of the gene, with highest activity near a series of 221-bp repeat units in the middle of the gene. Regions outside the A gene are inactive.

Physical and physiological components of the graviresponses of wild-type and mutant Paramecium Tetraurelia

2000 ◽  
Vol 203 (6) ◽  
pp. 1059-1070 ◽  
Author(s):  
U. Nagel ◽  
H. Machemer
Keyword(s):  
Swimming Speed ◽  
Sedimentation Rates ◽  
Paramecium Tetraurelia ◽  
Wild Type ◽  
Centre Of Gravity ◽  
In The Wild ◽  
Type Population ◽  
G Value ◽  
Mutant Cells

Wild-type and the morphological mutant kin 241 of Paramecium tetraurelia showed improved orientation away from the centre of gravity (negative gravitaxis) when accelerations were increased from 1 to 7 g. Gravitaxis was more pronounced in the mutant. A correlation between the efficiency of orientation and the applied g value suggests a physical basis for gravitaxis. Transiently enhanced rates of reversal of the swimming direction coincided with transiently enhanced gravitaxis because reversals occurred more often in downward swimmers than in upward swimmers. The results provide evidence of a physiological modulation of gravitaxis by means of the randomizing effect of depolarization-dependent swimming reversals. Gravity bimodally altered propulsion rates of wild-type P. tetraurelia so that sedimentation was partly antagonized in upward and downward swimmers (negative gravikinesis). In the mutant, only increases in propulsion were observed, although the orientation-dependent sensitivity of the gravikinetic response was the same as in the wild-type population. Observed swimming speed and sedimentation rates in the wild-type and mutant cells were linearly related to acceleration, allowing the determination of gravikinesis as a linear (and so far non-saturating) function of gravity.

Energy deprivation and a deficiency in downstream metabolic target genes during the onset of embryonic heart failure in RXRalpha−/− embryos

Development ◽  
1998 ◽  
Vol 125 (3) ◽  
pp. 533-544 ◽  
Author(s):  
P. Ruiz-Lozano ◽  
S.M. Smith ◽  
G. Perkins ◽  
S.W. Kubalak ◽  
G.R. Boss ◽  
...  
Keyword(s):  
Target Genes ◽  
Subtractive Hybridization ◽  
Causative Factor ◽  
Mutant Mice ◽  
Intermediary Metabolism ◽  
Wild Type ◽  
Energy Deficiency ◽  
Downstream Target ◽  
Ultrastructural Studies ◽  
Energy Deprivation

RXRalpha null mutant mice display ocular and cardiac malformations, liver developmental delay, and die from cardiac failure around embryonic day (E) 14.5 pc. To dissect the molecular basis of the RXRalpha-associated cardiomyopathy, we performed subtractive hybridization and systematically characterized putative downstream target genes that were selectively lacking in the mutant embryos, both at early (E10.5) and late (E13.5) stages of mouse embryonic development. Approximately 50% of the subtracted clones (61/115) encoded proteins involved in intermediary metabolism and electron transport, suggesting an energy deficiency in the RXRalpha−/− embryos. In particular, clone G1, which encodes subunit 14.5b of the NADH-ubiquinone dehydrogenase complex, displayed a dose-dependent expression in the wild-type, heterozygous and RXRalpha mutant mice. This gene was also downregulated in a retinoid-deficient rat embryo model. ATP content and medium Acyl-CoA dehydrogenase mRNA were lower in RXRalpha mutant hearts compared to wild-type mice. Ultrastructural studies showed that the density of mitochondria per myocyte was higher in the RXRalpha mutant compared to wild-type littermates. We propose a model whereby defects in intermediary metabolism may be a causative factor of the RXRalpha−/− phenotype and resembles an embryonic form of dilated cardiomyopathy.

Murein components rescue developmental sporulation of Myxococcus xanthus

1982 ◽  
Vol 152 (1) ◽  
pp. 462-470 ◽  
Author(s):  
L J Shimkets ◽  
D Kaiser
Keyword(s):  
Fruiting Body ◽  
High Cell Density ◽  
Myxococcus Xanthus ◽  
Sodium Dodecyl ◽  
Protein Composition ◽  
Diaminopimelic Acid ◽  
Nutrient Level ◽  
High Cell ◽  
Wild Type ◽  
Nutrient Rich Medium

Murein (peptidoglycan) components are able to rescue sporulation in certain sporulation-defective mutants of Myxococcus xanthus. N-Acetylglucosamine, N-acetylmuramic acid, diaminopimelic acid, and D-alanine each increase the number of spores produced by SpoC mutants. When all four components are included they have a synergistic effect, raising the number of spores produced by SpoC mutants to the wild-type level. Murein-rescued spores are resistant to heat and sonic oscillation and germinate when plated on a nutrient-rich medium. They appear to be identical to fruiting body spores in their ultrastructure, in their protein composition, and in their resistance to boiling sodium dodecyl sulfate. Murein rescue of sporulation, like fruiting body sporulation, requires high cell density, a low nutrient level, and a solid surface.

Developmentally controlled telomere addition in wild-type and mutant paramecia

1988 ◽  
Vol 8 (1) ◽  
pp. 251-258
Author(s):  
J D Forney ◽  
E H Blackburn
Keyword(s):  
Surface Antigen ◽  
Base Pair ◽  
Gene Locus ◽  
Gene Sequence ◽  
Genetic Locus ◽  
Single Site ◽  
Paramecium Tetraurelia ◽  
Wild Type ◽  
Telomeric Sequences ◽  
Antigen Gene

We analyzed sites of macronuclear telomere addition at a single genetic locus in Paramecium tetraurelia. We showed that in homozygous wild-type cells, differential genomic processing during macronuclear development resulted in the A surface antigen gene being located 8, 13, or 26 kilobases upstream from a macronuclear telomere. We describe variable rearrangements that occurred at the telomere 8 kilobases from the A gene. A mutant (d48) that forms a telomere near the 5' end of the A gene was also analyzed. This mutant was shown to create simple terminal deletions; telomeric repeats were added directly to the truncated wild-type A gene sequence. In both the mutant and wild-type cells, the telomeric sequences (a mixture of C4A2 and C3A3 repeats) were added to various sequences within a specific 200- to 500-base-pair region rather than to a single site. No similarities were found in the primary sequences surrounding the telomere addition sites. The mutation in d48 changed the region of telomere addition at the A gene locus; this is the first example in ciliates of a mutation that affects the site of telomere addition.

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