Identification of a novel family of nonclassic yeast phosphatidylinositol transfer proteins whose function modulates phospholipase D activity and Sec14p-independent cell growth.

Yeast phosphatidylinositol transfer protein (Sec14p) is essential for Golgi function and cell viability. We now report a characterization of five yeast SFH (Sec Fourteen Homologue) proteins that share 24-65% primary sequence identity with Sec14p. We show that Sfh1p, which shares 64% primary sequence identity with Sec14p, is nonfunctional as a Sec14p in vivo or in vitro. Yet, SFH proteins sharing low primary sequence similarity with Sec14p (i.e., Sfh2p, Sfh3p, Sfh4p, and Sfh5p) represent novel phosphatidylinositol transfer proteins (PITPs) that exhibit phosphatidylinositol- but not phosphatidylcholine-transfer activity in vitro. Moreover, increased expression of Sfh2p, Sfh4p, or Sfh5p rescues sec14-associated growth and secretory defects in a phospholipase D (PLD)-sensitive manner. Several independent lines of evidence further demonstrate that SFH PITPs are collectively required for efficient activation of PLD in vegetative cells. These include a collective requirement for SFH proteins in Sec14p-independent cell growth and in optimal activation of PLD in Sec14p-deficient cells. Consistent with these findings, Sfh2p colocalizes with PLD in endosomal compartments. The data indicate that SFH gene products cooperate with "bypass-Sec14p" mutations and PLD in a complex interaction through which yeast can adapt to loss of the essential function of Sec14p. These findings expand the physiological repertoire of PITP function in yeast and provide the first in vivo demonstration of a role for specific PITPs in stimulating activation of PLD.

Transcriptional activity of Drosophila melanogaster ecdysone receptor isoforms and ultraspiracle in Saccharomyces cerevisiae.

The Drosophila melanogaster ecdysone receptor (EcR) is produced in three isoforms, which mediate developmental processes such as metamorphosis. These isoforms were expressed in Saccharomyces cerevisiae to elucidate aspects of receptor transcription activity in a highly defined genetic model system. All three EcR isoforms showed ligand-independent transcriptional activation of an ecdysone reporter gene and the amount of activation correlated with the size of the N-terminal A/B (transactivation) domain present in the isoform: EcR-B1>EcR-A>>EcR-B2. Upon co-expression with ultraspiracle (Usp), transcriptional activation was further increased with EcR-B1 or EcR-A, but was unchanged with EcR-B2 or a truncated EcR lacking the A/B N-terminal domain (EcRDeltaA/B). Thus, the enhanced activity from Usp may depend on the presence of an N-terminal domain of EcR. Co-expression with Usp of several chimeric receptors of the EcR and the mouse androgen receptor (mAR) identified one chimera, composed of the mAR N-terminus and the remainder from EcR (mAR¿EcR-CDEF) that was transcriptionally silent and inducible by Usp. In contrast, the vertebrate homologue, human retinoic acid receptor (RXRalpha), showed ligand-independent transcription when co-expressed with EcRDeltaA/B but not mAR¿ EcR-CDEF. Therefore, RXRalpha does not require its partner to possess an N-terminal domain, yet is intolerant of a heterologous N-terminus. Similarly, the human vitamin D receptor, which has a short N-terminal region, showed greater ligand-independent transcription in the presence of RXRalpha than in the presence of Usp. These results reveal a mechanistic basis for the differential activities among the EcR isoforms, and between Usp and RXRalpha. Furthermore, they provided the foundation for a genetic screen to identify potential insecticides as well as accessory proteins for Usp and EcR.

Cell wall active antifungal compounds produced by the marine fungus Hypoxylon oceanicum LL-15G256. III. Biological properties of 15G256 gamma.

15G256 gamma is a cyclic lipopeptide antifungal agent discovered in a mechanism of action screen for cell wall acting antifungal agents. The compound shows moderate activity in both greenhouse tests against plant disease caused by pathogenic fungi and in in vitro tests against human fungal pathogens. Microscopic examination of treated fungi suggests that the compound acts by the inhibition of cell wall biosynthesis. However, in vitro inhibition of Neurospora crassa glucan and chitin synthase were only observed at high drug concentrations suggesting that 15G256 gamma may act on a novel cell wall target.

Multiple copies of PBS2, MHP1 or LRE1 produce glucanase resistance and other cell wall effects in Saccharomyces cerevisiae.

Five sequences were isolated by selection for multiple copy plasmids that conferred resistance to laminarinase, an enzyme that specifically degrades cell wall beta(1-3) glucan linkages. Strains carrying three of these plasmids showed alterations in cell wall glucan labelling. One of these plasmids carried PBS2, a previously identified, non-essential gene which produces a variety of phenotypes and encodes a mitogen-activated protein kinase kinase analogue (Boguslawski and Polazzi, 1987). Cells carrying PBS2 at multiple copy show a small decrease in cell wall beta(1-6) glucans. Measurements of beta(1-3) glucan synthase activity in multi-copy PBS2 cells showed an approximate 30-45% increase in enzyme specific activity while a pbs2 delta disruption strain showed a decrease in glucan synthase activity of approximately 45% relative to control. A pbs2 delta disruption strain was laminarinase super-sensitive and supersensitive to K1 killer toxin while a strain carrying PBS2 at multiple copy was resistant to killer toxin. A second plasmid carried a portion of the MHP1 gene which has been reported to encode a microtubule-interacting protein (Irminger-Finger et al., 1996). The MHP1 gene product is a predicted 1398 amino acid protein and only approximately 80% of the amino portion of this protein is required for laminarinase resistance. Cells carrying the amino portion of MHP1 at multiple copy show a decrease in high molecular weight cell wall beta(1-6) glucans and were killer toxin resistant while a disruption strain was viable and killer toxin super-sensitive. Cells carrying this plasmid showed decreased levels of high molecular weight beta(1-6) glucans and increased glucan synthase activity. The laminarinase resistance conferred by the third plasmid mapped to the previously uncharacterized YCL051W open reading frame and this gene was therefore named LRE1 (laminarinase resistance). The LRE1 gene encodes a non-essential 604 amino acid hydrophilic protein. Unexpectedly, cells carrying LRE1 at multiple copy show no alteration in cell wall glucans or glucan synthase activity. Subcloning experiments demonstrated that the production of these cell wall effects requires the presence of both LRE1 and YCL052C (PBN1), a second open reading frame present on the original plasmid. Cells carrying multiple copies of PBN1 alone show no significant alterations in cell wall glucans or glucan synthase activity, indicating that these effects require the presence of multiple copies of both genes.

Induction signals for vancomycin resistance encoded by the vanA gene cluster in Enterococcus faecium.

The induction of vancomycin resistance in enterococci containing the vanA gene cluster is thought to be controlled by a two-component sensor-response regulator system encoded by vanR and vanS. Eight inducing compounds were identified by screening a panel of more than 6,800 antibiotics and synthetic compounds including the three tested glycopeptides (vancomycin, avoparcin, and ristocetin), two other cell wall biosynthesis inhibitors (moenomycin and bacitracin), two cyclic peptide antibiotics (antibiotic AO341 beta and polymyxin B), and a macrocyclic lactone antibiotic (moxidectin). Induction activity by structurally unrelated antibiotics suggests that the induction signal is not a structural feature of vancomycin.

Cloning of the late genes in the ergosterol biosynthetic pathway of Saccharomyces cerevisiae--a review.

Research on the ergosterol biosynthetic pathway in fungi has focused on the identification of the specific sterol structure required for normal membrane structure and function and for completion of the cell cycle. The pathway and its end product are also the targets for a number of antifungal drugs. Identification of essential steps in ergo-sterol biosynthesis could provide new targets for the development of novel therapeutic agents. Nine of the eleven genes in the portion of the pathway committed exclusively to ergosterol biosynthesis have been cloned, and their essentiality for aerobic growth has been determined. The first three genes, ERG9 (squalene synthase), ERG1 (squalene epoxidase), and ERG7 (lanosterol synthase), have been cloned and found to be essential for aerobic viability since their absence would result in the cell being unable to synthesize a sterol molecule. The remaining eight genes encode enzymes which metabolize the first sterol, lanosterol, to ultimately form ergosterol. The two earliest genes, ERG11 (lanosterol demethylase) and ERG24 (C-14 reductase), have been cloned and found to be essential for aerobic growth but are suppressed by mutations in the C-5 desaturase (ERG3) gene and fen1 and fen2 mutations, respectively. The remaining cloned genes, ERG6 (C-24 methylase), ERG2 (D8AE7 isomerase), ERG3 (C-5 desaturase), and ERG4 (C-24(28) reductase), have been found to be nonessential. The remaining genes not yet cloned are the C-4 demethylase and the C-22 desaturase (ERG5).

Nikkomycin Z is a specific inhibitor of Saccharomyces cerevisiae chitin synthase isozyme Chs3 in vitro and in vivo.

Nikkomycin Z inhibits chitin synthase in vitro but does not exhibit antifungal activity against many pathogens. Assays of chitin synthase isozymes and growth assays with isozyme mutants were used to demonstrate that nikkomycin Z is a selective inhibitor of chitin synthase 3. The resistance of chitin synthase 2 to nikkomycin Z in vitro is likely responsible for the poor activity of this antibiotic against Saccharomyces cerevisiae.

Identification of a gene encoding a new Ypt/Rab-like monomeric G-protein in Saccharomyces cerevisiae.

A Saccharomyces cerevisiae sequence cloned by serendipity was found to encode a protein that is a new member of the Ypt/Rab monomeric G-protein family. This sequence shows high homology to the yeast genes SEC4 and YPT1 and, like SEC4 and YPT1, is essential for viability. The sequence was localized to chromosome V based upon hybridization to pulse-field gel-separated yeast chromosomes. The sequence has been deposited in the GenBank data library under Accession Number L17070.

The identification of a gene family in the Saccharomyces cerevisiae ergosterol biosynthesis pathway.

The Saccharomyces cerevisiae ERG24 gene, encoding sterol delta 14 reductase (Erg24p), was cloned by selecting strains carrying sequences on a 2 mu-based vector for resistance to the morpholine fungicide, fenpropimorph (Fp). Four distinct plasmid inserts which conferred Fp resistance (FpR) were recovered (plasmids pML99, pML100, pML101 and pM103). Although Fp is reported to inhibit activity of Erg24p and sterol delta 8-delta 7 isomerase (Erg2p; encoded by ERG2), none of the inserts had restriction maps resembling ERG2. In addition, a 2 mu plasmid overexpression of the ERG2 sequence did not produce FpR. Characterization studies were focused on plasmid pML100, because it was the only plasmid to confer FpR consistently when tested in a number of different genetic backgrounds. Tests with a panel of fungicides indicated that pML100 conferred significant resistance only to compounds (Fp, tridemorph, fenpropidin and azasterol) which have a shared site of action, Erg24p. An insertional disruption of pML100 resulted in an obligate anaerobic phenotype, indicating a lesion in sterol biosynthesis. Sterol analysis of the disrupted mutant demonstrated the accumulation of ignosterol, indicating a loss of Erg24p activity. A SphI-XbaI fragment of pML100 was sequenced, revealing the presence of an ORF encoding a 438-amino-acid protein, which is highly similar to those encoded by two previously reported yeast drug sensitivity genes, sts1+ (Schizosaccharomyces pombe) and YGL022 (S. cerevisiae). Analyses of these genes demonstrated that strains carrying disruptions of sts1+ or YGL022 have ergosterol biosynthesis defects in the enzyme, sterol C-24(28) reductase (Erg4p; encoded by ERG4).(ABSTRACT TRUNCATED AT 250 WORDS)

Development of improved cell-based assays and screens in Saccharomyces through the combination of molecular and classical genetics.

Traditionally, the discovery of pharmaceutical and agrochemical products has largely depended on mass screening. Over the years, screen design and screening programs have evolved in terms of the sensitivity with which active material can be identified, the number of samples that can be tested, and the types of molecular targets and cellular functions that can be conveniently assayed. More recently, screens with desirable properties have been developed for a great variety of molecular targets through the exploitation of Saccharomyces molecular biology and genetics. Recent advances have enabled researchers to develop yeast-based screens for agents acting on a number of new therapeutic targets: G-protein linked receptors, cytoplasmic receptors, ion (potassium) channels, novel fungal cell wall enzymes, fungal sterol biosynthesis enzymes, antiviral targets, immunosuppressive targets, cyclic nucleotide phosphodiesterase, oncogenes and the multiple drug resistance (MDR) protein.

Brefeldin A causes a defect in secretion in Saccharomyces cerevisiae.

Brefeldin A (BFA) blocks secretion in mammalian cells and causes the redistribution of Golgi resident membrane proteins to the endoplasmic reticulum (Klausner, R. D., Donaldson, J. G., and Lippincott-Schwartz, J. (1992) J. Cell Biol. 116, 1071-1080). The target(s) of BFA and its mechanism of action remain unknown. The yeast Saccharomyces cerevisiae represents an ideal organism in which to identify the BFA targets, since many molecules essential for vesicular traffic have been already identified taking advantage of the powerful genetics of this system. Unfortunately, wild type S. cerevisiae strains are largely insensitive to BFA (Hayashi, T., Takatsuki, A., and Tamura, G. (1982) Agric. Biol. Chem. 46, 2241-2248). Here we demonstrate that an erg6 mutant (Gaber, R., Copple, D., Kennedy, B., Vidal, M., and Bard, M. (1989) Mol. Cell. Biol. 9, 3447-3456) defective in the biosynthesis of ergosterol is sensitive to BFA. Treatment of erg6 cells with BFA results in an arrest in growth and causes a block in secretion similar to that seen in mammalian cells treated with BFA. Our data suggest that the changes in the erg6 strain allows BFA entry and that this strain can be used to examine the molecular mechanism of BFA action.

Dactylocyclines, novel tetracycline derivatives produced by a Dactylosporangium sp. I. Taxonomy, production, isolation and biological activity.

A screen for antibiotics with activity against tetracycline-resistant microorganisms has led to the isolation of Dactylosporangium sp. (ATCC 53693), a producer of several novel tetracycline derivatives. The major fermentation products, dactylocyclines A and B, were purified and MIC values determined against tetracycline-resistant and tetracycline-sensitive Gram-positive bacteria. The dactylocyclines represent the first naturally occurring tetracycline C2 amides which lack cross resistance with tetracycline.

Lanomycin and glucolanomycin, antifungal agents produced by Pycnidiophora dispersa. I. Discovery, isolation and biological activity.

The antifungal agents lanomycin and glucolanomycin were isolated from Pycnidiophora dispersa. The compounds were active against species of Candida and dermatophytes but were inactive against Aspergillus fumigatus and Gram-positive and Gram-negative bacteria. The compounds inhibited the cytochrome P-450 enzyme lanosterol 14 alpha-demethylase, and are believed, therefore, to have a mode of action similar to the azole and bis-triazole class of antifungal agents.

Pathogenicity of Candida albicans auxotrophic mutants in experimental infections.

Auxotrophic and prototrophic control strain pairs of Candida albicans constructed by molecular biology methodologies were evaluated for pathogenicity in a systemic mouse model. Mutants that were auxotrophic for adenine, uracil, and heme each showed a lowered level of pathogenicity relative to control strains. It can be concluded from these experiments that decreased pathogenicity in each case is due to the auxotrophic mutation, because mutant and control strains were constructed so as to differ at a single locus. These observations suggest that new therapeutic agents for Candida infections might be designed based upon the inhibition of biosynthetic pathways that, in some cases, might be absent from the host.

The use of beta-galactosidase gene fusions to screen for antibacterial antibiotics.

The desirable features for a screening assay to detect antibacterial antibiotics include 1) high specificity for the desired antibiotic type 2) high sensitivity 3) lack of interference by other compounds likely to be associated with the antibiotic of interest and 4) ease of operation to allow a large number of samples to be tested. These characteristics are largely found in screens employing strains carrying fusions between antibiotic induced promoters and the structural genes for Escherichia coli beta-galactosidase. Screens were designed based upon fusions with three antibiotic induced promoters: the tetracycline induced tetA/tetR promoter from transposon Tn10, the erythromycin induced promoter from the Staphylococcus aureus ermC erythromycin-resistance gene and the chloramphenicol induced promoter from the S. aureus cat86 chloramphenicol-resistance gene. Because there have been no reports of vancomycin induced resistance determinants, a Tn903 random gene fusion pool was screened to isolate a vancomycin induced gene fusion. This gene fusion was induced fairly specifically by glycopeptide antibiotics and the fusion was used as the basis for a glycopeptide screen.

Candida albicans in patients with the acquired immunodeficiency syndrome: absence of a novel of hypervirulent strain.

To determine whether Candida albicans in patients with AIDS represents a unique strain, C. albicans isolated from 24 patients with AIDS was compared with Candida isolated from 23 healthy adults. Resistance to 5-fluorocytosine, synthesis of amino acids and nucleotides, sugar use, and enzyme activity patterns were similar among isolates from the two groups. Molecular analysis revealed similar banding patterns of EcoRI restriction fragments of DNA between 2.5-3 and 6-7 kb. In addition, the frequency of a dimorphic 3.7-versus 4.2-kb band, identified by agarose gel electrophoresis and by probing Southern transfers of EcoRI digests with a cloned fragment of C. albicans DNA encoding 25S ribosomal RNA, was not significantly different between the AIDS-derived and control C. albicans. C. albicans isolated at different time points in the course of disease and from different sites in individual patients showed identical DNA fingerprints. The similarity in isolates of C. albicans that cause disease in AIDS patients and those present in healthy subjects suggests that the candidiasis associated with AIDS is not due to the presence of a unique or particularly virulent strain but is likely the consequence of a defect in host defense mechanisms.

Cloning and characterization of the 2,3-oxidosqualene cyclase-coding gene of Candida albicans.

2,3-Oxidosqualene (OS) cyclase (OSC) catalyzes the conversion of OS to lanosterol, an essential step in the biosynthesis of sterols. The Candida albicans gene (ERG7) encoding OSC was cloned by complementation of a Saccharomyces cerevisiae OSC mutant (erg7). Two different Erg+ clones were isolated that contain a common overlapping region. The minimum region required for complementation was determined to be approx. 3.2 kb and a single 2.7-kb ERG7 transcript was detected. The cloned Candida ERG7 DNA complemented an additional nonconditional erg7 allele and a temperature-sensitive erg7 mutation. OSC activity was restored in the mutants as determined by [14C]acetate incorporation in vivo as well as incorporation in vitro in cell-free extracts using either [14C]isopentenyl pyrophosphate or [3H]OS as substrate. The level of OSC produced from expression of a single copy of the Candida ERG7 sequence was sufficient to allow growth of the S. cerevisiae erg7 mutants in the absence of exogenous ergosterol. These data support the contention that the Candida ERG7 sequence is the structural gene for OSC.