Gametes of the unicellular green alga Chlamydomonas reinhardtii undergo sexual adhesion via enormous chimeric Hyp-rich glycoproteins (HRGPs), the plus and minus sexual agglutinins, that are displayed on their flagellar membrane surfaces. We have previously purified the agglutinins and analyzed their structural organization using electron microscopy. We report here the cloning and sequencing of the Sag1 and Sad1 genes that encode the two agglutinins and relate their derived amino acid sequences and predicted secondary structure to the morphology of the purified proteins. Both agglutinin proteins are organized into three distinct domains: a head, a shaft in a polyproline II configuration, and an N-terminal domain. The plus and minus heads are related in overall organization but poorly conserved in sequence except for two regions of predicted hydrophobic a-helix. The shafts contain numerous repeats of the PPSPX motif previously identified in Gp1, a cell wall HRGP. We propose that the head domains engage in autolectin associations with the distal termini of their own shafts and suggest ways that adhesion may involve head-head interactions, exolectin interactions between the heads and shafts of opposite type, and antiparallel shaft-shaft interactions mediated by carbohydrates displayed in polyproline II configurations.

The timely breakdown of the extracellular matrix by proteolytic enzymes is essential for development, morphogenesis and cell proliferation in plant and animal cells. Sporangin of the unicellular green alga Chlamydomonas reinhardtii that mediates breakdown of the sporangial cell wall to liberate the daughter cells after cell division is characterized as a subtilase-like serine protease. The sporangin gene is specifi cally transcribed during S/M phase in a synchronized vegetative cell cycle. In immunoblot analyses using a polyclonal antibody raised against the sporangin polypeptide, the enzyme is synthesized after mitotic cell division and accumulated in the daughter cells before hatching. Immunofl uorescence analyses showed that sporangin is localized to the fl agella of the daughter cells within the sporangial cell wall, and released into the culture medium. The data suggest that sporangin is released from fl agella concurrently with the digestion of sporangial cell wall, and then the daughter cells are hatched from the sporangia in the Chlamydomonas vegetative cell cycle. Abbreviations: ECM , extracellular matrix ; EST , expressed sequence tag ; GST , glutathione S -transferase ; PBS , phosphatebuffered saline ; RACE , rapid amplifi cation of cDNA ends .The nucleotide sequence of sporangin cDNA has been deposited in the EMBL/Genbank database under the accession number AB303361.

Cells of Chlamydomonas reinhardtii undergo gametogenesis to produce sexually competent gametes under nitrogen-starved conditions. By using a synchronized system for gametogenesis of early G1 cells, several previously identified marker genes and 18 novel nitrogen-starved gametogenesis (NSG) genes isolated by macroarray analysis were placed into at least three temporal classes of expression. Early genes are induced transiently in the first 2 h after transfer to nitrogen-free medium. Middle genes are strongly induced between 3 h and 4 h after nitrogen removal, a time corresponding to the acquisition of mating competency, suggesting their involvement in the gamete program. Late genes are induced between 5 h and 8 h after nitrogen removal, a time after the completion of gametic differentiation, suggesting that they are not directly involved in the formation of sexually competent gametes. All of the 18 NSG genes examined are induced in both mating-type plus and minus gametes and about two-thirds of the genes are also expressed in the mitotic cell cycle, especially at S/M phases.

When mating type plus and minus gametes of Chlamydomonas are mixed, they agglutinate with each other via their flagella, fuse, then initiate the zygote formation program which includes synthesis of the zygote cell wall, fusion of nuclei and chloroplasts, and the digestion of chloroplast DNA from the minus parent. The mRNAs from gamete and zygote cells was isolated and hybridized to cDNA-macroarray filters both to identify new genes expressed during the mating reaction and the early zygote formation process and to analyze the gene expression programs that underlie these sexual processes. Twenty-one novel genes were identified in this screen, designated as EZY (early zygote expressed) genes. The EZY genes included genes encoding proteins whose function is unknown, and genes encoding proteins that appear to be involved in processes such as cell wall synthesis, gene expression, intracellular trafficking or secretion, and vesicular transport in zygotic cells. All of the EZY genes were strongly induced within 1 h during the mating process, including early zygote formation. The transcriptional characteristics of EZY genes were analyzed by using the fusion-defective mutant fus mt(+). Among the EZY genes, 12 genes were not activated in fusion-defective conditions, suggesting that cell fusion is required for their expression. The remaining nine that were transcribed in fusion-defective fus matings were also inducible by cell wall removal in either vegetative or gametic cells, indicating that these genes were induced only indirectly by the cAMP signaling pathway initiated by flagellar agglutination as a result of mating-induced cell wall loss.