Disruption-deletion cassettes are powerful tools used to study gene function in many organisms, including Saccharomyces cerevisiae. Perhaps the most widely useful of these are the heterologous dominant drug resistance cassettes, which use antibiotic resistance genes from bacteria and fungi as selectable markers. We have created three new dominant drug resistance cassettes by replacing the kanamycin resistance (kan(r)) open reading frame from the kanMX3 and kanMX4 disruption-deletion cassettes (Wach et al., 1994) with open reading frames conferring resistance to the antibiotics hygromycin B (hph), nourseothricin (nat) and bialaphos (pat). The new cassettes, pAG25 (natMX4), pAG29 (patMX4), pAG31 (patMX3), pAG32 (hphMX4), pAG34 (hphMX3) and pAG35 (natMX3), are cloned into pFA6, and so are in all other respects identical to pFA6-kanMX3 and pFA6-kanMX4. Most tools and techniques used with the kanMX plasmids can also be used with the hph, nat and patMX containing plasmids. These new heterologous dominant drug resistance cassettes have unique antibiotic resistance phenotypes and do not affect growth when inserted into the ho locus. These attributes make the cassettes ideally suited for creating S. cerevisiae strains with multiple mutations within a single strain.

We have combined techniques of genetics and histochemistry to identify genes required for the nucleocytoplasmic export of mRNA in the budding yeast Saccharomyces cerevisiae. We adapted in situ hybridization using a digoxigenin-labeled oligo(dT)s o probe to localize poly(A) + RNA in fixed yeast cells and used yeast strains carrying the rnal-1 mutation to develop an assay. The rnal-1 mutation is the only previously described mutation that causes defects in mRNA export. As visualized with this RNA localization assay, rnal-1 strains accumulated poly(A) + RNA at the nuclear periphery at the nonpermissive temperature. This was in contrast to the RNA localization pattern of wild-type cells or rnal-1 cells grown at permissive temperature. Wild-type cells showed bright uniform cytoplasmic staining with little detectable RNA in the nuclei. We used this RNA localization assay to screen a bank of temperature-sensitive yeast strains for mutants with inducible defects in mRNA trafficking. Strains identified in this manner are designated RAT mutants for ribonucleic acid trafficking. The ratl-1 allele conferred temperature-sensitive accumulation of poly(A) + RNA in one to several intranuclear spots that appear to lie at the nuclear periphery. RNA processing was unaffected in ratl-1 strains, except for an inducible defect in trimming the 5' end of the 5.8S rRNA. The wild-type RAT1 gene was cloned by complementation; it encodes an essential ll6-kD protein with regions of homology to the protein encoded by SEP1 (also known as DST2, XRN1, KEM1, and RARS). Seplp is a nucleic acid binding protein, a 5'---> 3' exonuclease, and catalyzes DNA strand transfer reactions in vitro. We discuss the possible significance of the Ratlp/Seplp homology for RNA trafficking. We also discuss the potential of this RNA localization assay to identify genes involved in nuclear structure and RNA metabolism.[Key Words: mRNA export; yeast; in situ hybridization; RAT1; RNA1; SEP1]

The immunosuppressants cyclosporin A (CsA) and FK506 inhibit the protein phosphatase calcineurin and block T-cell activation and transplant rejection. Calcineurin is conserved in microorganisms and plays a general role in stress survival. CsA and FK506 are toxic to several fungi, but the common human fungal pathogen Candida albicans is resistant. However, combination of either CsA or FK506 with the antifungal drug¯uconazole that perturbs synthesis of the membrane lipid ergosterol results in potent, synergistic fungicidal activity. Here we show that the C.albicans FK506 binding protein FKBP12 homolog is required for FK506 synergistic action with¯uconazole. A mutation in the calcineurin B regulatory subunit that confers dominant FK506 resistance (CNB1-1/CNB1) abolished FK506±¯uconazole synergism. Candida albicans mutants lacking calcineurin B (cnb1/cnb1) were found to be viable and markedly hypersensitive to¯u-conazole or membrane perturbation with SDS. FK506 was synergistic with¯uconazole against azole-resistant C.albicans mutants, against other Candida species, or when combined with different azoles. We propose that calcineurin is part of a membrane stress survival pathway that could be targeted for therapy. Keywords: calcineurin/Candida albicans/cyclosporin A/ uconazole/antifungal drugs IntroductionThe immunosuppressants cyclosporin A (CsA) and FK506 block rejection of transplanted organs by inhibiting signaling pathways required for T-cell activation (Schreiber and Crabtree, 1992). Both drugs are in widespread clinical use and have had a dramatic impact on transplant therapy. Interestingly, CsA and FK506 are natural products of soil-dwelling bacteria or fungi (reviewed in Cardenas et al., 1994). Both drugs are toxic to a variety of fungi, suggesting one natural role might be to inhibit competing microorganisms in the soil (Tropschug et al., 1989;Breuder et al., 1994;Odom et al., 1997a;Arndt et al., 1999). CsA and FK506 exert immunosuppressive and antifungal effects by inhibiting calcineurin (Liu et al., 1991;Foor et al., 1992;Nakamura et al., 1993;Breuder et al., 1994;Odom et al., 1997a;Fox et al., 2001), a conserved Ca 2+ -calmodulin activated protein phosphatase (reviewed in Klee et al., 1998;Hemenway and Heitman, 1999;Aramburu et al., 2000). Calcineurin is a heterodimer comprised of a catalytic A and a regulatory B subunit (Hubbard and Klee, 1989;Anglister et al., 1994;Watanabe et al., 1995). CsA and FK506 do not inhibit calcineurin on their own, but ®rst bind to small, abundant, conserved binding proteins (immunophilins). CsA associates with cyclophilin A, and FK506 with FKBP12, to form protein±drug complexes that inhibit calcineurin by binding to the interface between the A and B subunits (Haddy et al., 1992;Li and Handschumacher, 1993;Milan et al., 1994;Cardenas et al., 1995b;Grif®th et al., 1995;Kawamura and Su, 1995;Kissinger et al., 1995). CsA and FK506 target this unique region of calcineurin and do not inhibit other phosphatases.The mechanisms of action of CsA and FK506 are conserved from fungi to h...