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1.
Chlamydomonas reinhardii cells were broken in a French press and the soluble fraction was tested for agglutination activity. Deflagellated cell bodies ofmt + andmt - gametes yielded soluble fractions that were able to isoagglutinate gametes of the opposite mating type. When the wild-type gametes of opposite mating types were mixed, the cell body-agglutinins were used up during flagellar agglutination and subsequent cell fusion. When thefus mt + andmt - gametes agglutinated without successive fusion, the amount of cell body-agglutinins sharply decreased, then increased and reached the premixing level: the recovery was blocked by cycloheximide. When cells were treated with EDTA or trypsin, the cell body-agglutinins as well as flagellar surface-agglutinins were completely lost without apparent loss of motility. The EDTA extract contained the same amount of agglutinins as observed in the cell bodies before extraction, and this amount was about 100 times higher than that in the EDTA extract of isolated flagella. By the addition of trypsin inhibitor, the trypsinized gametes resynthesized the cell body-agglutinins. The process was sensitive to cycloheximide in both mating type gametes and to tunicamycin inmt + gametes.Abbreviations mt +/- mating type plus or minus - CHI cycloheximide - TI trypsin inhibitor - TM tunicamycin  相似文献   

2.
Gametes ofChlamydomonas eugametos produce membrane vesicles, called isoagglutinins, which are shed into the culture fluid. It is assumed that they originate from the flagellar membrane for, like flagella, they can bind to the flagellar surface of gametes of the opposite mating type (mt). The composition ofmt - isoagglutinin was investigated with respect to this agglutinability. When the agglutination factor present on the surface ofmt - isoagglutinins (PAS-1.2) was removed, together with other membrane bound glycoproteins, the membrane vesicles were rendered inactive. They could be reactivated however by incubation with the extracted glycoproteins in a time-and concentration-dependent manner. The agglutination factor proved to be necessary yet sufficient in itself for the reactivation process to occur. Experiments with CsCl density gradients showed that the agglutination factor truly bound to the vesicles during reactivation. Inactivated vesicles derived frommt + gametes could be reactivated to gainmt - properties. Reactivation was inhibited by prior treatment with trypsin. The results indicate that the agglutination factor inmt - isoagglutinins is an extrinsic membrane protein bound to an intrinsic proteinaceous receptor.Abbreviations GTC guanidine thiocyanate - mt +/- mating type plus or minus - PAS periodic acid Schiff  相似文献   

3.
A new study of sexual agglutination between Chlamydomonas eugametos gametes and between vis-à-vis pairs has been made using techniques that allow one to distinguish between the flagella or cell bodies of individual mating types (mt+ or mt-). It is shown that before mt+ and mt- gametes fuse in pairs, their flagella, which adhere over their whole length, are maintained in a particular conformation around the mt- cell body. In clumps of agglutinating gametes the cells are asymmetrically distributed with the mt+ gametes constituting the outer surface of the clumps with the mt- gametes on the inside. The flagella are then all directed towards the middle of the clump. This orientation of the flagella is maintained for approx. 8 min after cell fusion before the vis-à-vis pair becomes motile. At this stage, all the flagellar tips are activated. The original mt+ flagellar tips then deactivate and swimming is resumed. The original mt- flagella remain immotile and activated after cell fusion and eventually shorten by a third, but only 30 min or more after fusion. Motile vis-à-vis pairs eventually settle to the substrate when the gamete bodies fuse completely to form a zygote. Settling vis-à-vis pairs are attracted to those that have already settled, to glutaraldehyde-fixed pairs and to flagella isolated from mt- gametes. They are not chemotactically attracted, rather they are weakly agglutinated. Living vis-à-vis pairs can be shown to aggregate in rows with the cell bodies lying side by side. It is argued that the flagellar agglutination sites involved in gamete recognition are also involved in vis-à-vis pair aggregationAbbreviations mt+/- mating type plus or minus - FTA flagellar tip activation  相似文献   

4.
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  相似文献   

5.
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)  相似文献   

6.
The relative amounts of Concanavalin A (Con A) bound by gamete and vegetative flagella of both mating types (mt + and mt -) of Chlamydomonas eugametos were determined using 125I-Con A. Con A agglutinated all cell types by cross-linking their flagella in a random manner. No correlation was found between the extent of Con A-binding and Con A-mediated isoagglutination. Con A inhibited the sexual interaction between gametes at various levels. In mt + gametes it blocked sexual agglutination, whereas in mt - gametes it prevented papillar fusion. By SDS-gel electrophoresis nine Con A-binding components were found to be present in flagella. However, it was not possible to allocate a role in sexual agglutination to any of these components since they were present in all cell types, including vegetative cells which are not able to sexually agglutinate.Abbreviations Con A concanavalin A - SDS sodium dodecyl sulphate - TB Tris buffer - PBS phosphate buffered saline - HRP horse radish peroxidase - SEM scanning electron microscope - PAS periodic acid Schiff  相似文献   

7.
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.  相似文献   

8.
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  相似文献   

9.
Chlamydomonas eugametos gametes agglutinate sexually by their flagellar surfaces. The agglutination factor on mating type minus (mt-) gametes is thought to be a glycoprotein named PAS-1.2. To test this idea, an antiserum was raised against purified PAS-1.2., which reacted with isolated PAS-1.2 (immunoprecipitation tests) and blocked the ability of isolated PAS-1.2 to induce sexual twitching in mt + gametes. When tested with living cells, the antiserum specifically agglutinated mt - gametes and induced a reaction resembling twitching. Mt + flagella were shown to bind the antiserum (indirect immunofluorescence) but much less than mt - gametes. Mt - gametes pretreated with Fab fragments of the antiserum were unable to reproduce sexually, while treated mt + gametes were unaffected. This effect presumably results from the ability of the serum to block mt - sexual agglutination, for mt - isoagglutinin was completely inactivated by the serum, while mt + isoagglutinin was unaffected. It is therefore argued that PAS-1.2 is the in vivo mt - agglutination factor. However it is shown that the antiserum was able to react in vitro not only with PAS-1.2 but with several other proteins in both mt - and mt + flagella.Abbreviations SDS sodium dodecyl sulphate - PAS periodic acid-Schiff - GTC guanidine thiocyanate - mt +/- mating type plus or minus - PBS phosphate buffer-saline - Fab univalent antibody fragment The investigations were supported by the Foundation for Fundamental Biological Research (BION), which is subsidized by the Netherlands Organization for the Advancement of Pure Research (Z.W.O.)  相似文献   

10.
The effect of disulfide-reducing agent dithiothreitol (DTT) on the plus and minus agglutinins ofChlamydomonas reinhardtii gametes was studied. Live gametes of mating-type plus (mt +) lost their flagellar agglutinability by DTT treatment without any loss of cell motility and concurrently released into the medium agglutinin in an inactive form. DTT treated cells also lost completely their cell body-agglutinin. By contrast, the mating-type minus (mt -) gametes neither lost their agglutinability nor released agglutinin into the medium by DTT even at very high concentrations. In vitro experiments showed that plus agglutinin in solution is just as sensitive as that in vivo to DTT, whereas minus agglutinin is totally insensitive, and the sulfhydryl-oxidizing agent diamide restores the plus agglutinin activity immediately and completely. Isolated flagella from themt + gametes were also inactivated by DTT, but they retained the inactivated agglutinin on the surfaces. The results indicate that plus agglutinin, but not minus agglutinin, possesses disulfide bonds which are essential for the recognition/adhesion activity.Abbreviations mt +/- mating-type plus or minus - DTT dithiothreitol  相似文献   

11.
Behaviors of the flagellar apparatuses (flagella, basal bodies, microtubular roots, etc.), mating structures and eyespots of gametes during the fertilization of Monostroma nitidum were studied using field emission scanning electron microscopy and transmission electron microscopy. The biflagellate isogamete (mt+ and mt?) mating structure has a position that is converse between mt+ and mt? gametes relative to the flagellar beat plane and the eyespot. After the adhesion of mt+ and mt? gametes, gamete fusion occurred between the two mating structures. The cell fusion plane expanded to the cell surface as circumscribed by 1s–2d roots in mt+ gamete and 1d–2s roots in the mt? gamete. Two sets of flagellar apparatuses lay side by side in the planozygote and soon become mutually close. The no. 1 basal body of mt+ gamete and the no. 2 basal body of mt? gamete rotated in a counterclockwise direction, as viewed from the cell anterior. Then, the no. 2 basal body of mt+ gamete and the no. 1 basal body of mt? gamete slid into a face to face position. Finally, four flagella and basal bodies exhibited a cruciate arrangement. The basal bodies of the opposing pair (no. 1 and no. 2) were offset in a counterclockwise orientation by the basal body diameter. The 1s and 2d roots of the mt+ gamete lay nearly parallel to the 1d and 2s roots of the mt? gamete, respectively, at the cell fusion plane. Because of the asymmetric localization of the mating structure, association, and subsequent rearrangement of basal bodies and microtubular roots, two eyespots lay on the same side of the planozygote. After the settlement of the planozygote, the flagellar apparatus started to disintegrate in the zygote cytoplasm.  相似文献   

12.
Isolated flagella from gametes of both mating types (mt+ and mt-) of Chlamydomonas reinhardii were suspended in buffer containing 7% sucrose. After mixing instantaneous agglutination occurred, giving rise to clumps which seem to be stable for at least 24 h. Control experiments show that no aggregates are formed when gametic flagella of one mating type are mixed with flagella prepared from vegetative cells of the other mating type.This in vitro agglutination is inhibited by a number of salt solutions in the same concentration range in which the agglutination of live gametes is affected. Moreover the clumps of flagella tend to disaggregate completely when the salt solutions are added after agglutination has occurred, or by treatment with trypsin. These observations suggest that the in vitro agglutination of isolated gametic flagella indeed reflects their physiological role in the recognition step of the mating process, which appears to be possible without participation of live gametes.We have also investigated the activity of glycosyl transferases on isolated gametic flagella before and during the in vitro agglutination reaction. As there was no detectable increase in the activity of glycosyl transferases, our results do not favour the hypothesis that these enzymes are involved in the primary step of recognition between gametic flagella.Dedicated to Prof. Dr. Otto Kandler on the occasion of his 60th birthday  相似文献   

13.
Summary The studies on the kinetics of nucleoid destruction reported here showed that destruction of chloroplast nucleoids (ct nucleoids) of male origin began to occur at about 30 minutes after mixing of male (mt) and female (mt+) gametes. The timing of initiation of the destruction differed among zygotes but usually occurred during 50–120 minutes after mixing. About 10 minutes was required for complete digestion of the ct nucleoids. UV irradiation on young zygotes or addition of an RNA-synthesis inhibitor, actinomycin D, to the incubation medium during the first 0–30 minutes after mixing almost completely inhibited the incorporation of3H uridine into the cell nuclei and the preferential destruction without inhibiting cell nuclear fusion. These results suggest that soon after mating,de novo RNA synthesis is concerned for the preferential destruction of ct nucleoids. To determine in which of the two cell nuclei in the zygotes the RNA is synthesized, each gamete (mt, mt+) was irradiated with UV and mated with unirradiated gametes of opposite mating type. This treatment of the male gametes had no effect on the incorporation of3H uridine into cell nuclei and the preferential destruction of ct nucleoids but UV irradiation of female gametes almost completely inhibited the incorporation of3H uridine into cell nuclei and the preferential destruction of ct nucleoids. Similar phenomena occurred in other crosses. The UV effect was photoreactivated in about 50% by white light, suggesting that the UV target is DNA. Thus, RNA synthesized in the cell nucleus of female origin soon after mating may be responsible for the preferential destruction of ct nucleoids of male origin  相似文献   

14.
Effects of tunicamycin (TM) on the sexual agglutinability and zygote formation of Saccharomyces cerevisiae were studied using the two kinds of haploid strains, inducible and constitutive for sexual agglutinability. Induction of sexual agglutinability by opposite mating type sex pheromone of inducible strains was inhibited by TM in mating type but not in a mating type. The recovery by temperature-shift-down from the temperature-suppressed sexual agglutinability of constitutive strains was enhanced by TM in a mating type but rather inhibited in mating type. Pretreatment with TM of constitutive strains enhanced sexual agglutinability in a mating type but not in mating type. The above-mentioned a-mating-type-specific agglutinability-enhancing actions of TM were discussed in relation to the action mechanism of pheromone which induces or enhances the sexual agglutinability of a cells.Zygote formation was inhibited by TM in both constitutive and inducible strains at concentrations which showed only partially inhibitory effect on sexual agglutinability.Abbreviations AI agglutination index - TM tunicamycin  相似文献   

15.
It has been previously shown that mild trypsinization of Chlamydomonas gametes reversibly inhibits steps of the mating process. Gametic agglutination is delayed 30–60 min, while cell wall hydrolysis and zygote formation are delayed 1–3 h. If gametes are pretreated with 5 μg/ml tunicamycin (TM) for 1 h and then trypsinized, the recovery of agglutination is blocked. These results indicate that N-glycosylated glycoproteins are involved in agglutination. Treatment of normal gametes with tunicamycin alone does not have a significant effect on agglutination and mating efficiency, suggesting that there is little or no turnover of the surface receptors before mating. Tunicamycin also interferes with cell growth and prevents the conversion of vegetative cells into gametes.  相似文献   

16.
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.  相似文献   

17.
Gametes of the marine green alga Ulva compressa L. are biflagellate and pear shaped, with one eyespot at the posterior end of the cell. The species is at an early evolutionary stage between isogamy and anisogamy. In the past, zygote formation of green algae was categorized solely by the relative sizes of gametes produced by two mating types (+ and ?). Recently, however, locations of cell fusion sites and/or mating structures of gametes have been observed to differ between mating types in several green algae (asymmetry of cell fusion site and/or mating structure positions). To use this asymmetry for determining gamete mating type, we explored a new method, field emission scanning electron microscopy (FE‐SEM), for visualizing the mating structure of U. compressa. When gametes were subjected to drying stress in the process of a conventional critical‐point‐drying method, a round structure was observed on the cell surfaces. In the mating type MGEC‐1 (mt+), this structure was located on the same side of the cell as the eyespot, whereas it was on the side opposite the eyespot in the mating type MGEC‐2 (mt?). The gametes fuse at the round structures. TEM showed an alignment of vesicles inside the cytoplasm directly below the round structures, which are indeed the mating structures. Serial sectioning and three‐dimensional construction of TEM micrographs confirmed the association of the mating structure with flagellar roots. The mating structure was associated with 1d root in the MGEC‐1 gamete but with 2d root in the MGEC‐2 gamete.  相似文献   

18.
Chlamydomonas eugametos gametes of opposite mating type make cell-cell contact via their flagellar surfaces. This contact triggers an increase in the intracellular level of cyclic AMP (cAMP) and several cellular responses which are necessary for cell fusion. Here, we show that wheat-germ agglutinin, which binds to the flagellar surface and induces all mating responses, also increased the intracellular cAMP level. Dibutyryl-cAMP added to non-mating gametes induced flagellar twitching, cell-wall lysis, mating-structure activation, flagellartip activation and an increase in agglutinability. It did not induce agglutinin transport to the flagellar tip (tipping) and may not be the direct cause of flagellar twitching and flagellar-tip activation. In non-illuminated cells, dibutyryl-cAMP was far more effective in evoking mating reactions than in illuminated cells. Light induced a 50% decrease in the cAMP level within 1 min. Adenylate cyclase was found to be associated with cell membranes but only 8% of the total was present in the gamete flagella.Abbreviations db-cAMP dibutyryl-cAMP - FTA flagellar tip activation - Mab monoclonal antibody - mt /mt+ mating-type minus/plus - WGA wheat-germ agglutinin We gratefully acknowledge the fruitful discussions with Dr. Rainer Gilles of the Department of Biochemistry at the University of Cologne (FRG), and the advice generously given by Dr. Roel van Driel of the Department of Biochemistry at the University of Amsterdam (The Netherlands).  相似文献   

19.
Summary The two interfertile algal species Chlamydomonas reinhardtii and C. smithii possess physically distinct mitochondrial (mit) genomes. Recently, use was made of this difference to demonstrate that sexual zygotes transmit the mit DNA from the mating-type minus (mt -, or paternal) parent exclusively. Diploid clones homozygous or heterozygous for the mt locus and carrying the mit genome of either of the two species were constructed by sexual crosses or artificially induced fusions. Haploid x diploid and diploid x diploid crosses were performed in order to analyze the role of both the mt locus and ploidy on the mode of transmission of mit DNA to the meiotic progeny. The inheritance of the mit DNA was determined by use of two molecular probes which hybridize to different regions of the organelle genomes. The mt u+/mt - gametes, which behave as mt - in the mating reaction, usually transmit their mit genome to the meiotic progeny, as do mt - or mt -/mt - gametes, regardless of the ploidy of the mt + gametes. In the cross mt + x mt +/mt - however, 2 zygospore clones (out of 14) transmitted recombinant DNA molecules containing a large segment of the C. reinhardtii mit genome and a 1 kb fragment typical of C. smithii. It can thus be concluded that, contrary to what was observed earlier for chloroplast gene transmission: (1) mt - is dominant to mt +with regard to mit DNA transmission, and (2) nuclear ploidy has little, if any, effect on mit DNA transmission.  相似文献   

20.
Sears BB  Boynton JE  Gillham NW 《Genetics》1980,96(1):95-114
In Chlamydomonas reinhardtii, gamete differentiation is induced by nitrogen deprivation. While cellular nitrogen content and amount of chloroplast DNA in cells of both mating types are reduced during gametogenesis, the spontaneous transmission of paternal (mt-) chloroplast alleles in crosses is specifically affected by the stringency of the nitrogen starvation regime used for pregrowth and gametogenesis of the mt- parent. In all cases, reciprocal crosses yielded biparental zygospores whose clones contain predominantly cells expressing only the chloroplast alleles from the maternal (mt+) parent. No differences attributable to strain divergence were seen in chloroplast gene inheritance pattern, DNA content, or the relative frequency of transmission of paternal chloroplast alleles to progeny of biparental zygospores.  相似文献   

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