Antheridiogen (A CE ) is a pheromone that is required for the development of male gametophytes in the homosporous fern Ceratopteris richardii . Subtractive hybridization of cDNAs isolated from A CE -treated and non-A CE -treated gametophytes was used to isolate genes that are induced by the pheromone. The expression of one gene, ANI1 (for antheridiogen induced), was induced within 3 hr of A CE treatment, but its expression was transient. Patterns of ANI1 expression in wild-type and mutant gametophytes show that ANI1 expression inversely correlates with the predicted activity of one of the sex-determining genes, TRANSFORMER5 ( TRA5 ). These data suggest that ANI1 transcription or transcript accumulation is directly or indirectly prevented by TRA5 in the absence of A CE and that A CE inactivates the TRA5 gene or its product, leading to the upregulation of ANI1 . Cycloheximide (no A CE ) induced the expression of ANI1 , also indicating that ANI1 expression is subject to negative regulation in the absence of A CE . The sequence and inferred protein structure of ANI1 suggest that it is a novel, extracellular protein. The secreted portion of the ANI1 protein potentially forms a ␤ barrel with superficial similarities to lipocalins, which bind small hydrophobic molecules such as pheromones, steroids, and odorants. ANI1 may be an extracellular carrier of A CE that is required to initiate the male program of development as the sexual fate of the young gametophyte is determined. INTRODUCTIONThe haploid gametophytes of many homosporous ferns, including Ceratopteris richardii , develop as either males or hermaphrodites (Figures 1A and 1B). The sex of the gametophyte is determined by the pheromone antheridiogen, or A CE in Ceratopteris (for antheridiogen Ceratopteris) (Schedlbauer and Klekowski, 1972;Banks et al., 1993). Ceratopteris spores develop as male gametophytes with numerous sperm-forming antheridia if they are continuously exposed to exogenous A CE from the time of spore germination. If A CE is removed from the medium surrounding an older male gametophyte, undifferentiated cells of the male prothallus will divide and differentiate a hermaphroditic prothallus, indicating that A CE is required to both initiate and maintain the male program of expression. In the absence of A CE , single spores develop as hermaphroditic gametophytes. These begin to produce and secrete A CE into the surrounding medium after they lose the ability to respond to the pheromone. The loss of sensitivity to A CE coincides with the development of a lateral, multicellular meristem, which is unique to the hermaphrodite ( Figure 1A) (Banks et al., 1993). Exogenous antheridiogen thus performs several functions in the young, sexually undetermined gametophyte: it promotes the differentiation of sperm-forming antheridia and simultaneously suppresses the development of the meristem, the egg-forming archegonia, and the synthesis of antheridiogen (Banks, 1997a).Although the structure of A CE is unknown, several lines of evidence suggest that A CE is a gibb...

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When cells of Proteus vulgaris were transferred from 37 to 42 C, a temperature at which they continue to grow almost optimally, they ceased to form flagella after approximately one generation time. This failure was due to a lesion in the flagellin-synthesizing process rather than the inability of these cells to assemble the organelle from constituents once formed. After transfer back to 37 C, these cells regained their ability to synthesize flagellin and form flagella, after one generation. When added during this period, chloramphenicol, rifampin, or penicillin prevented the synthesis of flagellin. The regeneration of the organelle at 37 C, then, requires growth for one generation, a period during which not only ribonucleic acid and protein synthesis, but also the presence of an intact cell envelope or concurrent synthesis of cell wall, are required.

Haloferax volcanii is to our knowledge the only prokaryote known to tolerate CRISPR-Cas mediated damage to its genome in the wild type background; the resulting cleavage of the genome is repaired by homologous recombination restoring the wild type version. In mutant Haloferax strains with enhanced self-targeting, cell fitness decreases and microhomology-mediated end joining becomes active, generating deletions in the targeted gene. Here we use self-targeting to investigate adaptation in H. volcanii CRISPR-Cas type I-B. We show that self-targeting and genome breakage events that are induced by self-targeting, such as those catalysed by active transposases, can generate DNA fragments that are used by the CRISPR-Cas adaptation machinery for integration into the CRISPR loci. Low cellular concentrations of self-targeting crRNAs resulted in acquisition of large numbers of spacers originating from the entire genomic DNA. In contrast, high concentrations of self-targeting crRNAs resulted in lower acquisition that was mostly centred around the targeting site. Furthermore, we observed naïve spacer acquisition at a low level in wild type Haloferax cells and with higher efficiency upon overexpression of the Cas proteins Cas1, Cas2 and Cas4. Taken together these findings indicate that naïve adaptation is a regulated process in H. volcanii that operates at low basal levels and is induced by DNA breaks.

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