Pleiotropic Mutations at the TUP1 Locus That Affect the Expression of Mating-Type-Dependent Functions in SACCHAROMYCES CEREVISIAE

The umr7–1 mutation, previously identified in a set of mutants that had been selected for defective UV-induced mutagenesis at CAN1, affects other cellular functions, including many of those regulated by the mating-type locus (MAT) in heterothallic Saccharomyces cerevisiae. The recessive umr7–1 allele, mapping approximately 20 cM distal to thr4 on chromosome III, causes clumpy growth in both a and α cells and has no apparent effect on a mating functions. However, α umr7 meiotic segregants fail to express several α-specific functions (e.g., high-frequency conjugation with a strains, secretion of the hormone α-factor and response to the hormone a-factor). In addition, α umr7 cells exhibit some a-specific characteristics, such as the barrier phenotype (Bar⁺) that prevents diffusion of α-factor and an increased mating frequency with α strains. The most striking property of α umr7 strains is their altered morphology, in which mitotic cells develop an asymmetric pear shape, like that of normal a cells induced to form "shmoos" by interaction with α-factor. Some a/α-specific diploid functions are also affected by umr7; instead of polar budding patterns, a/α umr7/umr7 diploids have medial budding like a/a, α/α and haploid strains. Moreover, a/α umr7/umr7 diploids have lost the ability to sporulate and are Bar⁺ like a or a/a strains. Revertant studies indicate that umr7–1 is a single point mutation. The umr7 mutant fails to complement mutants of both tup1 (selected for deoxythymidine monophosphate utilization) and cyc9 (selected for high iso-2-cytochrome c levels), and all three isolates have similar genetic and phenotypic properties. It is suggested that the product of this gene plays some common central role in the complex regulation of the expression of both MAT-dependent and MAT-independent functions.     

Synthesis of a-factor peptide from Saccharomyces cerevisiae and photoactive analogues via Fmoc solid phase methodology

a-Factor from Saccharomyces cerevisiae is a farnesylated dodecapeptide involved in mating. The molecule binds to a G-protein coupled receptor and hence serves as a simple system for studying the interactions between prenylated molecules and their cognate receptors. Here, we describe the preparation of a-factor and two photoactive analogues via Fmoc solid-phase peptide synthesis using hydrazinobenzoyl AM NovaGel™ resin; the structure of the synthetic a-factor was confirmed by MS-MS analysis and NMR; the structures of the analogues were confirmed by MS-MS analysis. Using a yeast growth arrest assay, the analogues were found to have activity comparable to a-factor itself.     

Development of a micelle-fractional precipitation hybrid process for the pre-purification of paclitaxel from plant cell cultures

A micelle-fractional precipitation hybrid process was developed for the effective pre-purification of the anticancer agent paclitaxel extracted from plant cell cultures. First, it was found that the efficiency of such a developed process could be remarkably enhanced by removing waxy substances originating from plant cells using the adsorbent sylopute. Paclitaxel yield was improved and the fractional precipitation time was shortened by increasing the surface area per working volume (S/V) of the reacting solution through the addition of a cation exchange resin (Amberlite IR120 or Amberlite 200), an anion exchange resin (Amberlite IRA400 or Amberlite IRA96), or glass beads. Most of the paclitaxel (>98%) could be obtained after about 12 h of fractional precipitation using Amberlite 200. Purity increased with increasing fractional precipitation time up to 9 h to about 85%, after which it showed little change. On the other hand, no paclitaxel precipitate was formed using either of the nonionic exchange resins because paclitaxel, which is hydrophobic, was strongly adsorbed on the hydrophobic resin surface. Since high-purity paclitaxel can be obtained in high yield and the precipitation time can be reduced by combining micelle formation with fractional precipitation, this hybrid method is expected to significantly enhance the final purification process.     

Mutants of SACCHAROMYCES CEREVISIAE Resistant to the α Mating-Type Factor

Mutants that are resistant to α-factor have been isolated from a mating-type haploid strains of yeast by direct selection on agar medium containing partially purified α-factor. All resistant mutants isolated were found to be sterile. They were characterized and compared with mutants previously isolated as nonmating. Among 93 able to mate at low frequency and to sporulate, none showed linkage to the mating-type locus. The results support the hypothesis that the response to α-factor by cells of mating-type a is essential for mating.     

Induction of sexual cell agglutinability of A mating type cells by alpha-factor in Saccharomyces cerevisiae.

Mating hormone, α-factor, which inhibits DNA synthesis and causes characteristic changes in cell morphology in $\text{a}$ mating type cells, was also responsible for induction of sexual cell agglutinability of $\text{a}$ mating type cells.     

Interconversion of Yeast Mating Types III. Action of the Homothallism (HO) Gene in Cells Homozygous for the Mating Type Locus

Mating type interconversion in homothallic Saccharomyces cerevisiae has been studied in diploids homozygous for the mating type locus produced by sporulation of a/a/a/α and a/a/α/α tetraploid strains. Mating type switches have been analyzed by techniques including direct observation of cells for changes in α-factor sensitivity. Another method of following mating type switching exploits the observation that a/α cells exhibit polar budding and a/a and α/α cells exhibit medial budding.—These studies indicate the following: (1) The allele conferring the homothallic life cycle (HO) is dominant to the allele conferring the heterothallic life cycle (ho). (2) The action of the HO gene is controlled by the mating type locus—active in a/a and α/α cells but not in a/α cells. (3) The HO (or HO-controlled) gene product can act independently on two mating type alleles located on separate chromosomes in the same nucleus. (4) A switch in mating type is observed in pairs of cells, each of which has the same change.     

A Novel Membrane-associated Metalloprotease, Ste24p, Is Required for the First Step of NH2-terminal Processing of the Yeast a-Factor Precursor

Many secreted bioactive signaling molecules, including the yeast mating pheromones a-factor and α-factor, are initially synthesized as precursors requiring multiple intracellular processing enzymes to generate their mature forms. To identify new gene products involved in the biogenesis of a-factor in Saccharomyces cerevisiae, we carried out a screen for MATa-specific, mating-defective mutants. We have identified a new mutant, ste24, in addition to previously known sterile mutants. During its biogenesis in a wild-type strain, the a-factor precursor undergoes a series of COOH-terminal CAAX modifications, two sequential NH₂-terminal cleavage events, and export from the cell. Identification of the a-factor biosynthetic intermediate that accumulates in the ste24 mutant revealed that STE24 is required for the first NH₂-terminal proteolytic processing event within the a-factor precursor, which takes place after COOH-terminal CAAX modification is complete. The STE24 gene product contains multiple predicted membrane spans, a zinc metalloprotease motif (HEXXH), and a COOH-terminal ER retrieval signal (KKXX). The HEXXH protease motif is critical for STE24 activity, since STE24 fails to function when conserved residues within this motif are mutated. The identification of Ste24p homologues in a diverse group of organisms, including Escherichia coli, Schizosaccharomyces pombe, Haemophilus influenzae, and Homo sapiens, indicates that Ste24p has been highly conserved throughout evolution. Ste24p and the proteins related to it define a new subfamily of proteins that are likely to function as intracellular, membrane-associated zinc metalloproteases.     

Interconversion of Yeast Mating Types I. Direct Observations of the Action of the Homothallism (HO) Gene

The HO gene promotes interconversion between a and α mating types. As a consequence, homothallic diploid cells are formed by mating between siblings descended from a single α HO or a HO spore. In order to determine the frequency and pattern of the mating-type switch, we have used a simple technique by which the mating phenotype can be assayed without losing the cell to the mating process itself. Specifically, we have performed pedigree analysis on descendants of single homothallic spores, testing these cells for sensitivity to α-factor.

The switch from α to a and vice versa is detectable after a minimum of two cell divisions. 50% of the clones tested showed switching by the four-cell stage. Of the four cells descended from a single cell, only the oldest cell and its immediate daughter are observed to change mating type. This pattern suggests that one event in the switching process has occurred in the first cell division cycle. Restriction of the switched mating-type to two particular cells may reflect the action of the homothallism system followed by nonrandom segregation of DNA strands in mitosis.

The mating behavior of cells which have sustained a change in mating type due to the HO gene is indistinguishable from that of heterothallic strains.

     

Novel reconstitution and enzymatic assay of the mitochondrial tricarboxylate carrier

The tricarboxylate carrier from beef liver mitochondria was reconstituted into liposomes using a protocol based on the absorption of Triton X-100 to hydrophobic Amberlite XAD-2 beads. The activity of the reconstituted carrier was determined spectroscopically by measuring the citrate/isocitrate exchange with an enzymatic assay. The Km for citrate obtained with this method was 35 microM and the Ki of 1,2,3-benzenetricarboxylate was 27 microM.     

The stimulation of anthraquinone production by Cinchona ledgeriana cultures with polymeric adsorbents

Summary Anthraquinones produced by suspension cultures of Cinchona ledgeriana are released into the medium, which becomes saturated with products late in the growth cycle. When a high affinity polymeric adsorbent, such as the macro-reticular Amberlite XAD-7, is added to the culture the concentration of anthraquinone in the medium is maintained at a low level and production may be stimulated 15-fold, yielding up to 20 mg/1/day. More than 90% of the product is released from the cells. For maximal yields it is shown that both the amount of adsorbent used and the time after sub-culture at which it is added to the system are critical. The value of such a method for product recovery from immobilised cells is discussed.     

Pre-treatment of growth medium with Amberlite® XAD-1180 produces higher levels of bacteriocin plantaricin 423

In this paper, we investigated the production of plantaricin 423, produced by Lactobacillus plantarum 423 in both MRS broth and partially deproteinated MRS with Amberlite XAD-1180. Improved production of plantaricin 423 was observed in MRS broth pre-treated with Amberlite XAD-1180. Precipitation of plantaricin with ammonium sulphate, followed by gel filtration chromatography and subsequent analysis on a modified tricine-SDS PAGE, significantly increased the yield and purity of plantaricin 423. This paper describes a novel method to partially deproteinate growth medium in order to facilitate bacteriocin purification.     

Enhanced anthraquinones production from adsorbent-treated Morinda elliptica cell suspension cultures in production medium strategy

Continuous removal of anthraquinones (AQ) by Amberlite polymeric adsorbents (XAD-4, XAD-7 and XAD-16) through in situ adsorption in Morinda elliptica cell suspension cultures is studied for product recovery and improvement of the overall titre. Ethanol was the best eluting solvent for effective recovery of AQ from all adsorbents. Pre-treatment of XAD-4 with sodium acetate not only enhanced intracellular AQ, but also AQ release and subsequent recovery from the adsorbent. The addition of sodium acetate pretreated XAD-4 on day 18 for 6-day contact period, achieved comparable cell growth to control (41 g/L), but with 1.3-fold higher intracellular AQ (124 mg/g DW) and two-fold increase in extracellular AQ (14.3 mg/L). High amount of adsorbent and longer contact period for the cultures entering stationary growth phase, stimulated AQ release and recovery but at the expense of cell growth. With 5–8.3 g XAD-4 adsorbent per litre M. elliptica culture in production (P) medium, between 60 and 90% AQ was recovered from extracellular AQ after 24–26 days of culture period.     

Control of yeast cell type by the mating type locus: II. Genetic interactions between MATα and unlinked α-specific STE genes

The alleles of the yeast mating type locus, MATα and MATa, determine the yeast cell types, a,α, and a/α. It has been proposed that the MATα2 product negatively regulates expression of unlinked a-specific genes, and that the MATα1 product positively regulates expression of unlinked α-specific genes. The behavior of mutants defective in MATα2, which are deficient in mating and in production of α-factor, can thus be attributed to antagonism between a-specific and α-specific functions expressed simultaneously in matα2^? strains. If this view is correct, then elimination by mutation of the specific functions required to mate as α may allow matα2 mutants to mate as a. In order to test this possibility, we examined the interactions between matα2 mutations and various unlinked mutations that cause α cells but not a cells to be mating defective (α-specific STE mutations). Three α-specific mutations (ste3, ste13 and kex2) were found to be non-allelic. Furthermore, although matα2 mutants mate weakly as a, matα2, ste3 double mutants, but not matα2 ste13 or matα2 kex2 double mutants, mate efficiently as a. The ability of matα2 ste3 strains to mate as a supports the view that matα2 mutants express a-specific mating functions, and suggests that a mating functions are expressed constitutively in MATa cells. The mating behaviour of the matα2 ste3 double mutant is consistent with the proposal that STE3 is positively regulated by the MATα1 product.     

Antimicrobial and cytotoxic activity produced by an isolate of the thermotolerant cyanobacterium (blue-green alga)Phormidium sp.

Summary Extracellular material from a thermotolerant isolate ofPhormidium sp. from the Azores was chromatographed using Amberlite XAD-7 in conjunction with C₁₈ Sep-Paks to reveal antimicrobial activity. Further examination indicated the possibility of anticancer activity, elucidated using a biochemical induction assay and the inhibition of the V79 cell line.     

Isolation and characterization of Saccharomyces cerevisiae mutants supersensitive to G1 arrest by the mating hormone a-factor

Summary Nine independent mutants which are supersensitive (ssl ^–) to G1 arrest by the mating hormone a-factor were isolated by screening mutagenized Saccharomyces cerevisiae MAT cells on solid medium for increased growth inhibition with a-factor. These mutants carried lesions in two complementation groups, ssl1 and ssl2. Mutations at the ssl1 locus were mating type specific: MAT ssl1 ^– cells were supersensitive to -factor but MAT ssl1 ^– were not supersensitive to -factor. In contrast, mutations at the ssl2. locus conferred supersensitivity to the mating hormone of the opposite mating type on both MAT, and MATa cells. The -cell specific capacity to inactivate externally added a-factor was shown to be lacking in MAT ssl1 ^– mutants whereas MAT ssl2. cells were able to inactivate a-factor. Complementation analysis showed that ssl2 and sst2, a mutation originally isolated as conferring supersensitivity to -factor to MATa cells, are lesions in the same gene. The ssl1 gene was mapped 30.5 centi-Morgans distal to ilv5 on chromosome XII.     

An improved procedure for the isolation of rat brown adipose tissue cells

A method for the isolation of brown adipocytes free of fat interferences and sensitive to noradrenalin is presented. The cells were isolated from pieces of brown adipose tissue with a collagenase treatment. The cells were obtained in the presence of heparin, in order to free the lipoprotein lipase attached to the cell surface. The cells were isolated in the presence of Amberlite XAD-2 [polymeric hydrophobic absorbent beads], which retained most of the fat droplets, formed from broken cells, during the process of disaggregation. The combined use of heparin and Amberlite XAD-2 during the isolation procedure resulted in a lowered cell basal oxygen consumption rate when compared with that of cells isolated with standard methods. The treatment presented lowered the availability of extracellular fatty acids for the isolated brown fat cells, resulting in lower operation of the thermogenin shunt, and thence in decreased basal oxygen consumption and higher sensitivity to glucose and noradrenalin stimulation.     

Metabolic products of microorganisms, 165th communication

Summary Isolation of siderochromes from culture filtrates by extraction with a mixture of chloroform and phenol was replaced by adsorption chromatography with Amberlite XAD-2. For the antibiotic albomycin, the purification was possible by ion-exchange chromatography with SP-Sephadex, and for the sideramine ferricrocin by exclusion chromatography with Bio-Gel P-2. Quantitation of albomycin was done by an agar plate diffusion test, and of ferricrocin by high-pressure liquid chromatography and photometric determination. Thinlayer and high-pressure liquid chromatographic systems were developed for checking homogeneity.Metabolic products of microorganisms. 164. W.A. König, C. Krauss, H. Zähner: 6-Chlor-Genistein und 6,3-Dichlor-Genistein. Helv. Chim. Acta in press     

Interconversion of Yeast Mating Types II. Restoration of Mating Ability to Sterile Mutants in Homothallic and Heterothallic Strains

The two mating types of the yeast Saccharomyces cerevisiae can be interconverted in both homothallic and heterothallic strains. Previous work indicates that all yeast cells contain the information to be both a and α and that the HO gene (in homothallic strains) promotes a change in mating type by causing a change at the mating type locus itself. In both heterothallic and homothallic strains, a defective α mating type locus can be converted to a functional a locus and subsequently to a functional α locus. In contrast, action of the HO gene does not restore mating ability to a strain defective in another gene for mating which is not at the mating type locus. These observations indicate that a yeast cell contains an additional copy (or copies) of α information, and lead to the "cassette" model for mating type interconversion. In this model, HMa and hmα loci are blocs of unexpressed α regulatory information, and HMα and hma loci are blocs of unexpressed a regulatory information. These blocs are silent because they lack an essential site for expression, and become active upon insertion of this information (or a copy of the information) into the mating type locus by action of the HO gene.     

Control of chemoselectivity of microbial reaction with resin adsorbent: enhancement of Baeyer–Villiger oxidation over reduction

Amberlite XAD-7, a hydrophobic polymer, was used to change microbial reaction of ketones from reduction to Baeyer–Villiger (BV) oxidation. Thus, D. magnusii NBRC 4600 and G. reessii NBRC 1112 could catalyze the BV reaction of ketones in the presence of the polymer while reduction of the substrates proceeded, and BV oxidation was scarcely found in the absence of the polymer.     

Immobilization of proteins on organic polymer beads

A new method is described for the immobilization of biologically active proteins onto several types of organic polymer beads. First, the soluble protein is modified by reaction with an excess of a hydrophobic imidoester, for example methyl 4-phenyl-butyrimidate, at ca. pH 9 and 0 degrees . Excess imidoester and side products resulting from imidoester hydrolysis are separated from the hydrophobic protein derivative by exclusion chromatography or dialysis. A suspension of polymer beads (e.g. Amberlite XAD-7) is then added to a solution of the modified protein at room temperature or below and stirred gently for 1-2 h. The polymer beads are allowed to settle, separated from the solution by decantation or filtration, washed, and resuspended in an appropriate buffer. Quantitative adsorption of protein to the polymer beads is observed under such conditions. The synthesis of seven hydrophobic imidoesters and their use for the immobilization of trypsin onto several types of porous polymer beads is described. The immobilizations of trypsin, yeast alcohol dehydrogenase, and E. coli asparaginase by this procedure with high recoveries of catalytic activity, suggests that it will be applicable to a large number of biologically active proteins.