Membrane glycoproteins of Chlamydomonas eugametos flagella

The flagellar glycoproteins exposed on Chlamydomonas eugametos gametes were labeled by means of lactoperoxidase, diiodosulfanilic acid and chloramine T, and characterised in SDS-electrophoresis gels. The medium from gamete cultures contains particles (isoagglutinins) that agglutinate gametes of the opposite mating type. When crude preparations of these particles were subjected to isopycnic centrifugation in a caesium chloride gradient, two bands of particles were found. The lighter, active band consisted of membrane vesicles. The denser, inactive band consisted of cell wall material. The active band had the same glycoprotein composition as membrane vesicles artificially made from isolated flagella. Preparations of glagella were also separated on a caesium chloride cushion into pure flagella and cell wall material. The flagella, but not the cell wall material, isoagglutinated opposite gametes. Again the glycoprotein composition of pure flagella was similar to that of pure isoagglutinin vesicles. No difference was detected between the protein and glycoprotein compositions of flagella and isoagglutinins from both mating types.Abbreviations LPO lactoperoxidase - PB phosphate buffer - DISA diazotized ¹²⁵I-iodo-sulfanilic acid - SDS sodium dodecyl sulphate - CBD coomassie Brilliant Blue - PAS periodic acid Schiff     

Sexual agglutination factor from Chlamydomonas eugametos

Chlamydomonas eugametos gametes can sexually agglutinate via their flagellar surfaces whereas vegetative cells cannot. Therefore, flagellar glycoproteins, present in gamete cells but absent from vegetative cells, were investigated as prospective mt ^-agglutination factors. They were identified as periodic acid Schiff (PAS) stained bands separated in sodium dodecyl sulphate-polyacrylamide electrophoresis gels. Gamete-specific bands were determined by comparison with equivalent gels of vegetative flagella and by immunological techniques using antisera raised against isolated mt ^- gamete flagella. Four high molecular weight flagellar glycoproteins proved to be gamete specific (PAS-1.2, PAS-1.3, PAS-3 and PAS-4). They were extracted from flagella by 3 M guanidine thiocyanate, separated in a column of Sepharose 2B, and tested for in vitro agglutination activity on mt ⁺ gametes. A single peak of activity was found to be correlated with the presence of the PAS-1.2 band. It is shown that mt ^- agglutination activity is related to the concentration of this glycoprotein in flagellar membranes.Abbreviations SDS sodium dodecyl sulphate - PAS periodic acid Schiff - GTC guanidine thiocyanate - mt ^-/+ mating type plus or minus     

Light dependence of sexual agglutinability in Chlamydomonas eugametos

The mating activity of mating-type plus gametes of Chlamydomonas eugametos depends on light. Cells lost their ability to agglutinate with mating-type minus gametes after a dark period of 30 min. They regained their agglutinability after 10 min exposure to light. Other mating reactions, such as tipping and flagellar tip activation, were not dependent upon light. Since cycloheximide and tunicamycin did not affect the light-induced activation of flagellar agglutinability, no protein synthesis or glycosylation is involved in this process. Equal amounts of biologically active agglutination factor could be extracted from cells placed either in light or in darkness. A minor portion of the active material was found to be located on the flagellar surface of illuminated cells. No active material was found on the flagellar surface of dark-exposed cells, whereas their cell bodies contained the same amount of active material as the cell bodies of illuminated cells. Since a light-induced flow of agglutination factors from the cell body to the flagella could not be detected and dark-exposed cells could be slightly activated by amputation or fixation by glutaraldehyde, we propose that light affects flagellar agglutinability by an in-situ modification of the agglutination factor on the flagella. When mt ⁺ and mt ^- strains were crossed and the progeny examined for light-sensitivity, it was apparent that this phenomenon is not mating type-linked.Abbreviations and symbols FTA flagellar tip activation - mt ^+/- mating type plus or minus - WGA wheat-germ agglutinin     

Actin in mating structures of Chlamydomonas eugametos gametes

The presence of actin in Chlamydomonas eugametos mating structures was studied using monoclonal anti-actin antibodies. Immunofluorescent labelling of mating gametes clearly stained their mating structures and this was confirmed at the electron microscope level by immunogold labelling of sections. Anti-actin labelling also strongly stained the flagella at the flagellar collar regions and weakly stained the rest of the flagella. Treatment of gametes with 6–8% ethanol induced mating structures which protruded as large 'balloons'. Balloons stained brilliantly with anti-actin antibodies and weakly with FITC-phalloidin, a fluorescent reagent that stains F-actin. Isolated mating structure balloons and flagella were analyzed using western blotting. A prominent 43 kDa band, co-migrating with actin in erythrocyte ghosts, reacted with anti-actin antibodies. The results indicate that actin is present in mating structures and flagella of both mating types of C, eugametos .     

An inhibitor of cell fusion in Chlamydomonas eugametos

By a short treatment with acid of mt ^- gametes of Chlamydomonas eugametos, a factor is released which prevents gametic cell fusion, without affecting the viability of the cells. It has a very rapid action. By means of scanning electron microscopy it is shown that the factor has no influence on flagellar adhesion nor on the formation of a plasma papilla by cells of either mating type, but that it specifically inhibits the fusion of these papillae. Evidence is presented suggesting that this inhibitor has a predominant effect on mt ⁺ gametes. In cell pairs which are blocked with respect to papillar fusion, no flagellar disengagement occurs, which indicates that loss of agglutinability is a direct consequence of cell fusion.     

Factors Affecting Immunogenic Activity of Mycobacterial Ribosomal and Ribonucleic Acid Preparations

By following careful procedures, mycobacterial ribosomal fractions and ribonucleic acid (RNA) prepared by ethyl alcohol precipitation were obtained which have immunogenic activities similar to the viable attenuated H37Ra cells of Mycobacterium tuberculosis from which they were obtained. This comparison was based on the amount of ribonucleic acid (RNA) present. These preparations consisted of approximately 63% RNA and 37% protein; no deoxyribonucleic acid or polysaccharide was detected by chemical tests. A high correlation was found between the immunogenic activity of a preparation and the per cent increase in hyperchromicity at 260 nm of a ribonuclease-hydrolyzed portion. Final concentrations of sodium dodecyl sulfate higher than 0.25% when used for the preparation of the ribosomal fractions and RNA resulted in significantly lower immune responses and greater variation between experiments. This was not related to the amount of protein present. The stability of the ribosomal and RNA preparations was tested under a variety of conditions. The need for a good protective adjuvant again was shown since mouse serum readily hydrolyzed the RNA. Equal immunity was obtained after immunization by the intraperitoneal and subcutaneous routes; however, no immune response was obtained when the intravenous route was used. Preliminary results with RNA prepared with phenol showed that it was more easily degraded during preparation. This resulted in a lower immune response than was obtained with the RNA prepared with ethyl alcohol.     

Action Spectrum of the Activity of Acifluorfen-methyl, a Diphenyl Ether Herbicide, in Chlamydomonas eugametos

Light is required for the herbicide activity of diphenyl ether herbicides. An action spectrum of acifluorfen-methyl activity with Chlamydomonas eugametos (Moewus) determined that cell death occurred at two peaks of light; 450 and 670 nanometers. These data indicate both chlorophyll and carotenoids, but not riboflavin, are involved in herbicide toxicity.     

Gamete activity in mating strains of Chlamydomonas eugametos

The gamete activity of compatible mating strains of the isogamous, heterothallic species Chlamydomonas eugametos was investigated. Gamete activity was optimum within 4 h after flooding of agar slants and was maintained over a 24-h period. When male and female mating strains were mixed in proportions of 1:4, 2:3, 1:1, 3:2, and 4:1, the results based on zygote yield, indicated the strains exhibited different degrees of gamete activity. The male strain consistently showed less gamete activity than the female strain in a variety of culture conditions.     

Agglutination factor in the cell body of Chlamydomonas eugametos

Chlamydomonas eugametos gametes agglutinate via the surfaces of their flagella. The mating-type minus (mt ^-) agglutination factor is a high-molecular-weight glycoprotein called PAS-1.2, present on the exterior surface of the flagellar membrane. During flagellar regeneration, mt ^- gametes were able to agglutinate as soon as the flagella protruded as short stumps. This was also observed when protein synthesis was blocked, indicating that gametes possess a pool of PAS-1.2. When the exterior surface of flagella-less gametes was extracted and the proteins were subjected to gel electrophoresis, large quantities of PAS-1.2 were detected. Using anti-PAS-1.2 serum, the presence of PAS-1.2-like material was visualized on the plasma membrane of mt ^- gamete cell bodies. By assaying the biological activity of extracts of the cell bodies and of isolated flagella, it was calculated that the plasma membrane of the cell bodies contains 25 times the activity present in the flagella and could, therefore, represent a large pool of mt ^- agglutination factor.     

The cell wall of Chlamydomonas eugametos. Immunological aspects

An antiserum was raised against the major cell wall glycoprotein of Chlamydomonas eugametos which after purification reacted specifically with all individual wall components but not with intact cell walls. The antigenic sites in intact walls appear to be cryptic but become exposed on partial enzymatic degradation or in situ during daughter-cell release when the insoluble component is digested. Using the antiserum as a specific label for cell walls in various stages of disintegration, cell wall digestion during asexual and sexual reproduction was studied. It is also shown that while cell wall material is associated with isolated flagella, it is not normally associated with the flagella of intact cells.     

Endogenous oscillator controls surface density of mating receptors in Chlamydomonas eugametos

Summary We describe a circadian rhythm in the surface density of receptors that play a dominant role in the mating process of the unicellular green alga Chlamydomonas eugametos.These receptors — called agglutinins — are large glycoproteins extrinsically bound to the membrane of gamete flagella. We found circadian fluctuations in their density. Since inhibition of protein synthesis affected the agglutinin density without a lag period at any time,we conclude that the density was dependent on de novo synthesis and that the fluctuations in density are caused by circadian oscillations in the rate of agglutinin synthesis. This phenomenon evidently underlies the pronounced endogenous rhythm in mating competence that we described previously (Demets et al. 1987). Finally, we speculate on the nature of the time keeping mechanism that is generating these rhythmic events.     

Occurrence of O-methylated sugars in surface glycoconjugates in Chlamydomonas eugametos

Previously, we have shown that the monomeric-sugar composition of cell-surface-associated glycoconjugates of two strains of Chlamydomonas eugametos, of different mating type, differs strikingly (Gerwig et al. 1984, Carbohydr. Res. 127, 245–251). Besides the common occurrence of various pentoses and hexoses, the glycoconjugates of one strain contain 4-O-methyl xylose, a 2-O-methyl pentose (probably 2-O-methyl arabinose) and 3-O-methyl galactose, whereas those of the other strain contain 6-O-methyl mannose and 3-O-methyl glucose. In order to investigate whether these differences are relevant to the mating process of this organism, the sugar composition of the sexual progeny of these strains was analyzed. The ability to produce 4-O-methyl xylose, 2-O-methyl pentose and 3-O-methyl galactose on the one hand, and the ability to produce 6-O-methyl mannose and 3-O-methyl glucose on the other hand, appear to be genetically linked. However, the ability to produce either set of O-methyl sugars was inherited independently of mating type. O-Methylated sugars do not occur in the cell wall of C. eugametos, or in the cell-free medium, but only in surface-membrane-associated glycoconjugates, extractable with salt or detergent solutions.Abbreviation mt ^+/- mating-type plus or minus     

Sex-specific binding and inactivation of agglutination factor in Chlamydomonas eugametos

Gametes of opposite mating type (mt ⁺ and mt ^-) of the green alga Chlamydomonas eugametos agglutinate via their flagella as a prelude to sexual fusion. To quantitate sexual agglutination, an in vitro assay has been developed using ³⁵S-labeled flagella and the isolated mt ^-agglutination factor. It is shown that not only isolated flagella, but also the mt ^-agglutination factor rapidly bind to the flagella of intact gametes of the opposite mating type. This confirms the role of the mt ^-agglutination factor in determining the sexual agglutinability of mt ^-gametes. As a function of binding, the agglutinative power of the flagella of both mating types is destroyed by a temperature-sensitive process. Likewise, the mt ^-agglutination factor can be completely inactivated.Abbreviations Mt ^+/- mating type plus or minus - PAS periodic-acid Schiff-reagent - Hepes 4-(2-hydroxyethyl)-1-piperazineethansulfonic acid - HMC buffer Hepes buffer (10 mM. pH 7.2, containing 1 mM MgCl₂ and 1 mM CaCl₂)     

Phototactic responses of Chlamydomonas eugametos gametes before and after fusion

The phototactic behavior of Chlamydomonas eugametos gametes and vis-à-vis pairs was quantitated using a fully automated, computer-controlled microvideo image analysis system. Two different mt- (mating type minus) and one mt+ (mating type plus) strain, together with the two combinations of pairs were studied. One mt- strain of dark-adapted gametes was non-phototactic while the others were positively phototactic at all effective intensities of white light. The mt+ strain exhibited one of the strongest positive responses that has so far been reported in algae (r-values greater than 0.7). After sexual fusion, the mt+ cell powers the swimming vis-à-vis pair. Its phototactic behavior reversed on fusion, with the pairs swimming away from all effective light intensities, irrespective of whether its partner was formerly phototactic or not. However, when adapted to the dark for an hour or more, vis-à-vis pairs swam positively to the light. The ecological consequence could be that pairs settle and develop into zygotes under intermediate light intensities or at light-dark interfaces.     

Ultrastructure of Chlamydomonas eugametos palmelloids induced by chloroplatinic acid treatment.

Palmelloids induced in the unicellular green alga Chlamydomonas eugametos by chloroplatinic acid treatment have been studied electron microscopically. Thin-sectioned specimens revealed the multilayer nature of the cell walls after second division within the palmelloid. Although synchrony in cell division is lost, to a certain degree, within the palmelloid, the cells themselves appeared normal, and the presence of normal flagellar structure was confirmed. The presence of the eyespot was observed at the optical level as well as in the freeze-etched specimens. The above results support the hypothesis that the palmelloid condition of Chlamydomonas eugametos induced by chloroplatinic acid is due to an abnormality in cell wall formation rather than flagellar malfunction or loss.     

Flagellar tip activation in vis-à-vis pairs of Chlamydomonas eugametos

An alteration of the form and ultrastructure of the tips of the flagella of Chlamydomonas eugametos, occurring during sexual agglutination, is shown to be persistent in the mt ^- flagella of the resulting vis-à-vis pairs. It is argued that this phenomenon is related to the lack of motility of mt ^- flagella in vis-à-vis pairs of this species.