Isolation and Structure Determination of Blepharismin, a Conjugation Initiating Gamone in the Ciliate
            <i>Blepharisma</i>

Kubôta, Takashi; Tokoroyama, Takashi; Tsukuda, Y.; Koyama, H.; Miyake, Akio

doi:10.1126/science.179.4071.400

Cited by 75 publications

(23 citation statements)

References 5 publications

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“…In normal ASW membrane potential is dependent on the external K+ concentration to a muLch greater extent in light than in darkness, as reported earlier (4). In the presence of DNP this disparity is greatly reduced, and membrane potentials both in the dark and in light show a strong dependence on the external K-tconcentration.…”

supporting

confidence: 77%

Blepharmone: A Conjugation-Inducing Glycoprotein in the Ciliate Blepharisma

Miyake

Beyer

1974

Science

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supporting

confidence: 77%

Blepharmone: A Conjugation-Inducing Glycoprotein in the Ciliate Blepharisma

Miyake

Beyer

1974

Science

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“…The second stage, costimulation, requires actual contact between the two mating types [Bruns and Palestine, 19751. Many attempts have been made to analyze the molecular mechanism of the mating reaction andior to determine the sex factors in ciliates [Kubota et al, 1973;Miyake and Beyer, 1974;Braun and Miyake, 1975;Miceli et al, 1983;Concetti et al, 1986;Luporini et al, 1986;Ortenzi et al, 19901, but little is known of the molecular events leading to pair formation in T. thermophila.…”

Section: Conjugation In the Ciliatementioning

confidence: 99%

Ciliary polypeptides and glycoconjugates of wild‐type and mutant Tetrahymena thermophila: Starved versus nonstarved

Cheng

Hufnagel

1992

Dev. Genet.

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To investigate the role of cilia in mating interactions of Tetrahymena thermophila, ciliary membrane-rich fractions were isolated from two wild-type strains, a non-discharge mucocyst mutant which possesses mating behavior similar to wild-type, and a mating mutant which is able to costimulate cells of complementary mating type but cannot enter into pair formation. In each case, proteins from the ciliary membrane-rich fractions of starved, mating-competent ("initiated") cells were compared with those from non-starved, mating-incompetent ("non-initiated") cells, by gel electrophoresis and lectin blotting. In stained gels, a 43 kDa polypeptide was reduced or absent in initiated cells but present in non-initiated cells, in all strains. In silver-stained gels, a 25 kDa polypeptide was present in all strains, both initiated and non-initiated. In blots probed with Con A-peroxidase, a 25 kDa glycoprotein was present in ciliary membrane fractions from non-initiated cells and absent in membranes of initiated cells of the two wild-type strains and the mucocyst mutant, but is present in initiated and non-initiated cells of the mating mutant (several hypotheses are presented to explain these findings). In addition, ciliary proteins of the mating mutant included at least two unique Con A-binding polypeptides. Our results support the idea that development of mating competence during starvation involves an extensive remodeling of ciliary membranes, and identify a 25 kDa glycoconjugate as having a potential role in control of pair formation during mating.

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“…In many cases, isolation of conjugation-inducing substances (gamones, mating pheromones, or mating substances) that induce mating-pair formation in conjugation is extremely difficult. In the ciliates, gamone 2 (blepharismone) in Blepharisma japonicum was the first identified conjugation-inducing substance (5). Thus far, in only three species, B. japonicum, Euplotes raikovi, and Euplotes octocarinatus, have the conjugation-inducing substances ever been isolated (see ref.…”

mentioning

confidence: 99%

Identification, characterization, and complete amino acid sequence of the conjugation-inducing glycoprotein (blepharmone) in the ciliate Blepharisma japonicum

Sugiura

Harumoto

2001

Proc. Natl. Acad. Sci. U.S.A.

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Conjugation in Blepharisma japonicum is induced by interaction between complementary mating-types I and II, which excrete blepharmone (gamone 1) and blepharismone (gamone 2), respectively. Gamone 1 transforms type II cells such that they can unite, and gamone 2 similarly transforms type I cells. Moreover, each gamone promotes the production of the other gamone. Gamone 2 has been identified as calcium-3-(2-formylamino-5-hydroxybenzoyl) lactate and has been synthesized chemically. Gamone 1 was isolated and characterized as a glycoprotein of 20 -30 kDa containing 175 amino acids and 6 sugars. However, the amino acid sequence and arrangement of sugars in this gamone are still unknown. To determine partial amino acid sequences of gamone 1, we established a method of isolation based on the finding that this glycoprotein can be concentrated by a Con A affinity column. Gamone 1 is extremely unstable and loses its biological activity once adsorbed to any of the columns that we tested. By using a Con A affinity column and native PAGE, we detected a 30-kDa protein corresponding to gamone 1 activity and determined the partial amino acid sequences of the four peptides. To isolate gamone 1 cDNA, we isolated mRNA from mating-type I cells stimulated by synthetic gamone 2 and then performed rapid amplification of cDNA ends procedures by using gene-specific primers and cloned cDNA of gamone 1. The cDNA sequence contains an ORF of 305 amino acids and codes a possibly novel protein. We also estimated the arrangement of sugars by comparing the affinity to various lectin columns.

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Isolation and Structure Determination of Blepharismin, a Conjugation Initiating Gamone in the Ciliate Blepharisma

Cited by 75 publications

References 5 publications

Blepharmone: A Conjugation-Inducing Glycoprotein in the Ciliate Blepharisma

Blepharmone: A Conjugation-Inducing Glycoprotein in the Ciliate Blepharisma

Ciliary polypeptides and glycoconjugates of wild‐type and mutant Tetrahymena thermophila: Starved versus nonstarved

Identification, characterization, and complete amino acid sequence of the conjugation-inducing glycoprotein (blepharmone) in the ciliate Blepharisma japonicum

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