The dimorphic yeast Yarrowia lipolytica is used as a model to study fungal differentiation because it grows as yeast-like cells or forms hyphal cells in response to changes in environmental conditions. Here, we report the isolation and characterization of a gene, ZNC1, involved in the dimorphic transition in Y. lipolytica. The ZNC1 gene encodes a 782 amino acid protein that contains a Zn(II)2C6 fungal-type zinc finger DNA-binding domain and a leucine zipper domain. ZNC1 transcription is elevated during yeast growth and decreases during the formation of mycelium. Cells in which ZNC1 has been deleted show increased hyphal cell formation. Znc1p-GFP localizes to the nucleus, but mutations within the leucine zipper domain of Znc1p, and to a lesser extent within the Zn(II)2C6 domain, result in a mislocalization of Znc1p to the cytoplasm. Microarrays comparing gene expression between znc1::URA3 and wild-type cells during both exponential growth and the induction of the yeast-to-hypha transition revealed 1,214 genes whose expression was changed by 2-fold or more under at least one of the conditions analyzed. Our results suggest that Znc1p acts as a transcription factor repressing hyphal cell formation and functions as part of a complex network regulating mycelial growth in Y. lipolytica.

The present study analyzed iNOS and eNOS mRNA expression and NO production during development of hepatic abscess caused by Entamoeba histolytica trophozoites. One 374-bp sequence, which displayed 88% identity to mammalian iNOS protein, was isolated from LPS-stimulated peritoneal hamster macrophages. A separate 365-bp cDNA sequence showed 99% identity with eNOS protein. iNOS mRNA was detected in hamsters during formation of amoebic liver abscesses, but not in control hamsters. eNOS mRNA expression was not modified. Serum nitrite concentration in hamsters infected with E. histolytica was 33 +/- 6 microM, in control hamsters was 20 +/- 3 microM. The study shows that iNOS mRNA expression and NO production are induced by E. histolytica trophozoites during amoebic liver abscess formation. However, in spite of iNOS mRNA expression and NO production, E. histolytica trophozoites induced liver abscess formation in hamster.

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Evidence has been obtained for the presence of the small 22 kDa GTP-binding Rho protein in dormant spores of Phycomyces blakesleeanus. lmmunoblotting with a polyclonal antibody against RhoA revealed a soluble and membraneassociated 22 kDa protein. When [32P]ADP-ribosylated by Clostridium botulinum C3 exotoxin the protein had a pl of 5-7, a value similar to that reported for other RhoA proteins. The 22 kDa protein was expressed a t all stages of growth investigated, but radiolabelling of the [32P]ADP-ribosylated protein increased when tube-formation occurred and decreased as the hyphae branched. Localization of RhoA during spore germination studied by immunofluorescence microscopy revealed the presence of RhoA in the cell membrane of the spore. When the spore started to swell, RhoA was observed as patches in the cell membrane which become concentrated in the neck region of the site of the protuberation tube, but this protein was never observed a t the point of growth of the hyphal tip. The above results suggest that RhoA associated with one or more membrane proteins could participate in the molecular mechanism involved in maintaining cell integrity during the extrusion of the germ-tube of P. blakesleeanus.

A DNA sequence homologous to a Gas DNA probe, and the corresponding Gas protein (stimulatory a-subunit of GTP-binding protein) were detected in PhyCOfVlYCeS b/akeS/eeanUS. The protein Was demonstrated in membrane fractions of dormant spores of this fungus using three different experimental approaches. Photoaff inity-labelling experiments with [a-3zP]GTP of the membrane fraction revealed two bands, of 56 and 32 kDa. The 56 kDa GTPbinding protein was detected by this method in all the stages of early development and growth investigated. Also, a spore protein of 56 kDa was ADP-ribosylated by cholera toxin, and a 56 kDa protein was detected by Western blotting with a specific antibody against mammalian Gas. These results indicate that Gas (56 kDa) is present in dormant spores of P. Makesleeanus. Using the ADP-ribosylation and Western blotting assays, Gas was detected during all stages of spore germination before the hyphae became highly branched, but it was not detected in the branched hyphae that formed 18 h after the initiation of spore germination. Therefore, Gas is expressed differentially during Phycomyces development. 36000