Abstract. Adhesion between Chlamydomonas reinhardtii gametes generates a rapid rise in cAMP levels which stimulates mating responses and zygotic cell fusion (Pasquale and Goodenough, 1987). We show here that sexual adhesion in vivo results in a twofold stimulation of flagellar adenylyl cyclase activity when the enzyme is subsequently assayed in vitro, a stimulation that is specifically blocked by Cd 2÷. A twofold stimulation is also elicited by the in vitro presentation of soluble cross-linking reagents (antisera and concanavalin A). In contrast, the 10-30-fold stimulation of the flagellar cyclase by in vitro exposure to 40°C, first described by Zhang et al. (1991), is insensitive to Cd 2÷ but sensitive to such drugs as trifluoperizine and dibucaine. The capacity for twofold stimulation is displayed by the vegetative and gametic enzymes but is lost when gametes fuse to form zygotes; in contrast, the 10-fold stimulation is displayed by the gametic and zygotic enzymes but not the vegetative enzyme. The signal-defective mutant imp-3 fails to generate the normal mating-triggered cAMP production and can be rescued by exogenous dibutyryl cAMP. It displays normal basal rates of flagellar cyclase activity and a normal twofold stimulation by sexual adhesion and by soluble cross-linkers, but it is defective in 40°C activation. The gametic cell-body adenylyl cyclase is stimulated when wild-type fagella, but not imp-3 flagella, undergo adhesive interactions in vivo, and it can be directly stimulated in vitro by cAMP presentation. We propose that the two levels of fagellar cyclase stimulation reflect either sequential steps in the activation of a single cyclase enzyme, with imp-3 blocked in the second step, or else the sequential activation of two different fagellar enzymes, with imp-3 defective in the second enzyme. We further propose that the cell-body enzyme is activated by the cAMP that is generated when flagellar cyclase activity is fully stimulated.

Six cDNA clones have been identified that are complementary to transcripts present in young zygotes of Chlamydomonas reinhardti but absent from vegetative and gametic cells. Five early transcripts are synthesized within 5 to 10 min of fertilization; the sixth, late, transcript is not synthesized until 90 min following fertilization. Synthesis of both classes requires cell fusion between gametes. Cycloheximide fails to inhibit early mRNA synthesis, indicating that transcription factors must preexist in the gametes and be activated by cytoplasmic confluence. By contrast, cycloheximide blocks synthesis of the late transcript, suggesting that an early protein product(s) is required for expression of the late gene. Restriction fragment length polymorphism analysis of inter-and intraspecific genetic crosses demonstrates that one of the early genes is very tightly linked to the mating-type locus.The single-celled alga Chlamydomonas reinhardtii undergoes an apparently simple type of fertilization and development (23, 24); gametes of opposite mating type (mt), called plus (mt+) and minus (mt-), fuse to form a diploid zygote, which quickly begins to synthesize a novel group of proteins, some of which are glycoproteins that go on to assemble into an insoluble wall around the cell (2, 18). In this article we report the cloning of six zygote-specific cDNAs, documenting that at least some of the observed fertilization-induced shifts in protein synthesis (18, 31) are due to novel gene expression in zygotes and not simply to the activation of maternal-mRNA equivalents. We found that five of the six genes are transcribed within 5 to 10 min after fertilization, whereas the sixth is not expressed until 90 min after fertilization; since the expression of the sixth is cycloheximide sensitive, we propose that a protein product of one or more of the early genes is required for the expression of such a late gene. The expression of the early genes precedes nuclear fusion and is cycloheximide insensitive, indicating that factors synthesized by the gametes must interact in the zygote to initiate the early program. Finally, we found that one of the five early genes was linked to the mating-type locus, which will make it possible to probe the genomic organization of this key regulatory element. MATERIALS AND METHODSCells. Except when designated, experiments were performed with strains CC620 (mt+) and CC621 (mt-). Vegetative cells were grown in liquid TAP medium (8a) and harvested during logarithmic growth; gametes were produced on TAP agar plates and suspended in N-free HSM (16). Equal numbers of mt+ and mt-gametes were mated at 2 x 107 to 5 x 107 cells per ml to produce zygotes. The efficiency of mating, determined as described previously (17), was generally 85 to 95%. Obvious cell wall secretion (as judged by adhesion of zygotes to one another and resistance to lysis with 1% Nonidet P-40) usually begins about 3 h after mating; however, this time has varied from 2.5 to 5 h in different experiments. * Corresponding author.DNA and RNA isolat...

Within the first hour of zygote maturation, Chlamydomonas reinhardtii cells stop synthesizing certain polypeptides that characterize the vegetative and gametic stages of the life cycle and initiate the synthesis of novel, zygote-specific polypeptides. At least six of these polypeptides are secreted into the medium, and fine-structural studies indicate that they represent components of the cell wall that is synthesized and secreted early in zygote development. We conclude that a new program of protein synthesis, and possibly also gene transcription, is initiated shortly after gametic cells fuse, a program that appears highly suited to celldifferentiation studies. KEY WORDS Chlamydomonaszygotes 9 secretion 9 cell differentiation During its simple life cycle, the unicellular eukaryotic protist Chlamydomonas reinhardtii passes through three states of differentiation: the haploid vegetative state, the haploid gametic state, and the diploid zygotic state. Gametogenesis is triggered by nitrogen starvation of vegetative cells (27) and requires about 12 h (17, 22). Zygotes form when gametes of opposite mating type (mr + and mr-) fuse to form quadriflagellated cells during the mating reaction, and early zygote development proceeds for about 24 h, during which time a thick zygote wall is elaborated (7).We wished to learn whether either of these lifecycle transitions was suitable for studies of eukaryotic cell differentiation, our criterion for suitability being that the transition should involve the production of readily detected, novel polypeptides whose synthesis is specific for a particular state of differentiation. We therefore began by subjecting cells at various stages of differentiation to 2-3-h pulses with [~4C]acetate, and analyzed the labeled polypeptides by slab-gel electrophoresis followed by autoradiography.When such experiments were performed with cells undergoing gametogenesis (24), all of the major polypeptides synthesized by gametes were found to have apparent counterparts in the vegetative-cell samples. The synthesis of many vegetative-cell polypeptides was observed to abate or cease entirely during gametogenesis so that shifts occurred in the relative intensities of various bands on the gels, but no major polypeptides specific to the gametic state could be distinguished by a variety of approaches.In contrast, we discovered that zygotic cell fusion in C. reinhardtii elicits the synthesis of a number of major zygotic polypeptides that are not evident in vegetative or gametic cells. Because the trigger for the onset of this "zygotic program" is gametic and probably nuclear fusion, the phenomenon is at least formally analogous to the trigger-J. CELL BIOLOGY 9 The Rockefeller University Press 9 0021-9525/78

A procedure is described for localizing antigen-antibody complexes in sodium dodecyl sulfate (SDS) polyacrylamide gels using 12~I-labeled protein A from Staphylococcus aureus. We use the procedure to probe antigenic cross-reactivities between Strongylocentrotus and Chlamydomonas o~-and fl-tubulins; we also demonstrate how the procedure can detect minor antibody species in an antiserum directed against a cell membrane. KEY WORDS lz~I-protein Aantigen-antibody complexes autoradiography tubulin membranesThe characterization of cellular components using immunological approaches has been hampered by the fact that many polypeptides of interest (e.g., tubulin, actin) are poor antigens, eliciting weak and/or nonprecipitating antisera that are often difficult to analyze. Moreover, many structures of interest (e.g., cell membranes) carry a complex array of components, some more antigenic than others. Antisera raised against intact membranes therefore contain a complex array of antibody species at varying titers, and such antisera have heretofore been of limited experimental value.We report here a simple, rapid, and highly sensitive method, which utilizes 125I-labeled protein A from Staphylococcus aureus for analyzing antisera against cellular components. We then show how the procedure can be applied to basic biological questions, presenting experiments which demonstrate interspecific relationships between a-and/3-tubulin polypeptides and experiments which reveal both the major and minor polypeptides of a cell surface membrane. MATERIALS AND METHODSProtein A (Pharmacia Inc., Piscataway, N. J.) was labeled with ~25I (New England Nuclear, Boston, Mass.) by the chloramine-T method of Greenwood et al. (5) except that the chloramine-T was added in four aliquots at 15-s intervals. The labeled product (sp act = 1.5 x 10 ~ cpm/tzg protein) was separated on a G-50 Sephadex column pre-run with 20 mg of bovine serum albumin. Greater than 60% of the radioactivity was covalently bound to protein A under these conditions, and recovery from the column was essentially complete.Sea urchin sperm tail axonemes and antitubulin antisera (raised in rabbits against vinblastine sulfate crystals of sea urchin [Strongylocentrotus] egg cytoplasmic tubulin [4]) were generous gifts of Dr. K. Fujiwara (Harvard Medical School, Boston, Mass.). Flagella were isolated and purified from Chlamydomonas reinhardi gametes by the pH-shock procedure of Witman et al. (12). Antiserum against C. reinhardi mt § gametic flagella (a-G +) was raised in a rabbit by standard procedures; details will be described elsewhere (U. W. Goodenough and D. Jurivich, J. Cell Biol., in press).Samples for gel electrophoresis were solubilized in lysis buffer [3% wt/vol sodium dodecyl sulfate (SDS), 5% vol/vol 2-mercaptoethanol, 10% glycerol, 0.01% wt/vol bromphenol blue, 6.25 mM Tris-HCl, pH 6.8) and boiled for 2 min before loading. Electrophoresis was carried out in slab gels (1 mm • 1 cm • 18 cm) as described in the figure legends, using the discontinuous buffer system of Lae...

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