The polarization of sterol- and sphingolipid-enriched domains (lipid rafts) has been linked to morphogenesis and cell movement in diverse cell types. In the yeast Saccharomyces cerevisiae , a dramatic polarization of sterol-rich domains to the shmoo tip was observed in pheromone-induced cells (M. Bagnat and K. Simons, Proc. Natl. Acad. Sci. USA 99: 14183-14188, 2002). We therefore examined whether plasma membrane lipid polarization contributes to the ability of the fungal pathogen Candida albicans to grow in a highly polarized manner to form hyphae. Interestingly, staining with filipin revealed that membrane sterols were highly polarized to the leading edge of growth during all stages of hyphal growth. Budding and pseudohyphal cells did not display polarized staining. Filipin staining was also enriched at septation sites in hyphae, where colocalization with septin proteins was observed, suggesting a role for the septins in forming a boundary domain. Actin appeared to play a role in sterol polarization and hyphal morphogenesis in that both were disrupted by low concentrations of latrunculin A that did not prevent budding. Furthermore, blocking either sphingolipid biosynthesis with myriocin or sterol biosynthesis with ketoconazole resulted in a loss of ergosterol polarization and caused abnormal hyphal morphogenesis, suggesting that lipid rafts are involved. Since hyphal growth is required for the full virulence of C. albicans , these results suggest that membrane polarization may contribute to the pathogenesis of this organism.

Two new yeast genes, ASF1 (Anti‐Silencing Function) and ASF2, as well as a C‐terminal fragment of SIR3, were identified as genes that derepressed the silent mating type loci when overexpressed. ASF2 overexpression caused a greater derepression than did ASF1. ASF1 overexpression also weakened repression of genes near telomeres, but, interestingly, ASF2 had no effect on telomeric silencing. Sequences of these two genes revealed open reading frames of 279 and 525 amino acids for ASF1 and ASF2, respectively. The ASF1 protein was evolutionarily conserved. MCB motifs, sequences commonly present upstream of genes transcribed specifically in S phase, were found in front of both genes, and, indeed, both genes were transcribed specifically in the S phase of the cell cycle. While an asf2 mutant was viable and had no obvious phenotypes, an asf1 mutant grew poorly. Neither mutant exhibited derepression of the silent mating type loci. The asf1 mutant was sensitive to methyl methane sulfonate, slightly UV‐sensitive and somewhat deficient in minichromosome maintenance. It also lowered the restrictive temperature of a cdc13^ts mutant. These phenotypes suggested a role for ASF1 in DNA repair and chromosome maintenance. The GenBank accession numbers for the ASF1 and ASF2 sequences are L07593 and L07649, respectively. © 1997 by John Wiley & Sons, Ltd.

The Philadelphia chromosome (Ph1), observed in greater than 90% of chronic myelogenous leukemia (CML) patients, results from a specific chromosomal translocation involving the c-abl gene. The translocation breakpoint occurs near c-abl and correlates with the production of an altered c-abl mRNA. In the CML-derived cell line K562, Ph1 is accompanied by a structurally altered c-abl protein (P210c-abl) with in vitro tyrosine kinase activity not detected with the normal c-abl protein (P145c-abl). We have examined c-abl proteins in other Ph1-positive CML cell lines and found that they all express P210c-abl. P210c-abl was also detected in bone marrow cells from CML patients with Ph1 in the accelerated and blast crisis phases of the disease. Comparison of the [35S]methionine-labeled tryptic peptides generated from the normal P145c-abl and P210c-abl showed that they have closely related structures, but additional polypeptide sequences are present in P210c-abl. Based on these results we propose that translocation of c-abl in Ph1-positive CML results in the creation of a chimeric gene leading to the production of a structurally altered c-abl protein with activated tyrosine kinase activity. The altered P210 c-abl protein is strongly implicated in the pathogenesis of CML.

The v-abl transforming protein P160v-abl and the P210c-abl gene product of the translocated c-abl gene in Philadelphia chromosome-positive chronic myelogenous leukemia cells have tyrosine-specific protein kinase activity. Under similar assay conditions the normal c-abl gene products, murine P150c-abl and human P145c-abl, lacked detectable kinase activity. Reaction conditions were modified to identify conditions which would permit the detection of c-abl tyrosine kinase activity. It was found that the Formalin-fixed Staphylococcus aureus formerly used for immunoprecipitation inhibits in vitro abl kinase activity. In addition, the sodium dodecyl sulfate and deoxycholate detergents formerly used in the cell lysis buffer were found to decrease recovered abl kinase activity. The discovery of assay conditions for c-abl kinase activity now makes it possible to compare P150c-abl and P145c-abl kinase activity with the altered abl proteins P160v-abl and P210c-abl. Although all of the abl proteins have in vitro tyrosine kinase activity, they differ in the way they utilize themselves as substrates in vitro. Comparison of in vitro and in vivo tyrosine phosphorylation sites of the abl proteins suggests that they function differently in vivo. The development of c-abl kinase assay conditions should be useful in elucidating c-abl function.