Genomic clones representing three Chlamydomonas reinhardtii genes homologous to the Drosophila hsp70 heat shock gene were isolated. The mRNAs of genes hsp68, hsp70, and hsp80 could be translated in vitro into proteins of Mr 68,000, 70,000, and 80,000, respectively. Transcription of these genes increased dramatically upon heat shock, and the corresponding mRNAs rapidly accumulated, reaching a peak at around 30 min after a shift to the elevated temperature. Light also induced the accumulation of the mRNAs encoded by these heat shock genes. A shift of dark-grown cells to light resulted in a drastic increase in mRNA levels, which reached a maximum at around 1 h after the shift. Thus, in Chlamydomonas, expression of hsp70-homologous heat shock genes appears to be regulated by thermal stress and light.
MATERIALS AND METHODSZygotes represent an important stage in the sexual cycle of the unicellular green alga Chlamydomonas relnhardtii. To study zygote germination at a molecular level, a protocol was elaborated for the generation of zygotes in large quanftites and a method was developed for the extraction from zygotes of RNA that could be translated in vitro.The sexual cycle of Chlamydomonas reinhardtii involves the fusion of two gametes of opposite mating type resulting in the formation of a diploid zygote. The mature zygote can subsequently be induced to undergo germination. After meiosis, four spores are released (9). The individual steps within this life cycle (all at a single cell level) represent defined stages in cell differentiation. The morphological and physiological characteristics of the individual cell types have been investigated in some detail (3,12). Although analyses of the mating reaction and zygote formation have provided insights into the mechanisms of cell-cell recognition (1, 18) and signal transduction ( 14), the cellular stages of zygote maturation and germination have so far not been analyzed at a molecular or biochemical level in C. reinhardtii. The reasons were the lack of procedures to obtain mature zygotes (i.e. zygotes able to germinate) in sufficient quantities and the lack of methods for the efficient disruption of these thick walled cells. Two major problems have previously prohibited the production of large numbers of zygotes. First, zygotes appear to require contact to a firm surface (e.g. an agar plate) for maturation. Zygotes left in liquid medium are different from plate matured zygotes (3,5). More important, zygotes from liquid medium germinate very poorly (3). Second, when newly formed zygotes are plated on agar plates for maturation, the mature zygotes become embedded within the agar and are difficult to recover. We describe here a novel method for the production of large numbers of mature zygotes that overcomes both of these problems, and we also present a reliable procedure for extraction of functional zygote RNA.I Supported by a grant of the Deutsche Forschungsgemeinschaft (SFB206). Strains and Culture ConditionsChlamydomonas reinhardtii wild-type strains 137c+ and 1 37c-(obtained from R. Matagne) were used for all experiments. One L cultures were inoculated from plates and grown in TAP2-medium (4) in 1 L round flasks with air bubbling and continuous illumination (30 uE m-2 s-') at 23C. Gametogenesis was induced by transferring vegetative cultures in midlog phase (cell density 1 -2 x 106 cells/mL) to nitrogen-free TAP-medium (16). Gametogenesis was performed under the conditions described above for the growth of vegetative cells. Production of Zygotes in Large QuantitiesEighteen to 20 h after induction of gametogenesis by nitrogen removal (cell density 4-8 x 106 cells/mL), equal numbers of mt+ and mt-gametes were mixed, usually 2 L of both. Portions (50-75 mL) of the mating mixture were then transferred to 100 mL Erlenmeyer flasks and incubated without shaking...
In the sexual life cycle of Chlamydomonas reinhardtii, three types of cells can be distinguished: vegetative cells, gametes, and zygotes. Using is vivo pulse labelling of proteins with 35S‐sulfate, followed by two‐dimensional separation of the polypeptides on polyacrylamide gels, we analyzed the patterns of protein synthesis typical for each cell type. Approximately 20% of the proteins detected were synthesized at very different rates when the pattern from vegetative cells was compared to that of gametes or zygotes. Gamete formation from vegetative cells is a two‐step process controlled by two extrinsic signals: first, nitrogen‐starvation induces the differentiation of vegetative cells to pregaroetes, which are then competent for a light–induced differentiation to mature gametes. The majority of the changes in rate of synthesis of different proteins during the switch from vegetative cells to gametes was observed already in pregametes. Between the stages of pregametes and gametes, changes in the rates of synthesis of four proteins were detected. Induction of zygote germination resulted in complex changes in the patterns of gene expression. Evidence is presented for three groups of proteins whose synthesis is turned on or turned off during zygote germination.
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