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.

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.

Chitinase and chitin synthase 1: counterbalancing activities in cell separation of Saccharomyces cerevisiae.

Previous results [E. Cabib, A. Sburlati, B. Bowers & S. J. Silverman (1989) Journal of Cell Biology 108, 1665-1672] strongly suggested that the lysis observed in daughter cells of Saccharomyces cerevisiae defective in chitin synthase 1 (Chs1) was caused by a chitinase that partially degrades the chitin septum in the process of cell separation. Consequently, it was proposed that in wild-type cells, Chs1 acts as a repair enzyme by replenishing chitin during cytokinesis. The chitinase requirement for lysis has been confirmed in two different ways: (a) demethylallosamidin, a more powerful chitinase inhibitor than the previously used allosamidin, is also a much better protector against lysis and (b) disruption of the chitinase gene in chs1 cells eliminates lysis. Reintroduction of a normal chitinase gene, by transformation of those cells with a suitable plasmid, restores lysis. The percentage of lysed cells in strains lacking Chs1 was not increased by elevating the chitinase level with high-copy-number plasmids carrying the hydrolase gene. Furthermore, the degree of lysis varied in different chs1 strains; lysis was abolished in chs1 mutants containing the scs1 suppressor. These results indicate that, in addition to chitinase, lysis requires other gene products that may become limiting.

The function of chitin synthases 2 and 3 in the Saccharomyces cerevisiae cell cycle.

The morphology of three Saccharomyces cerevisiae strains, all lacking chitin synthase 1 (Chs1) and two of them deficient in either Chs3 (calR1 mutation) or Chs2 was observed by light and electron microscopy. Cells deficient in Chs2 showed clumpy growth and aberrant shape and size. Their septa were very thick; the primary septum was absent. Staining with WGA-gold complexes revealed a diffuse distribution of chitin in the septum, whereas chitin was normally located at the neck between mother cell and bud and in the wall of mother cells. Strains deficient in Chs3 exhibited minor abnormalities in budding pattern and shape. Their septa were thin and trilaminar. Staining for chitin revealed a thin line of the polysaccharide along the primary septum; no chitin was present elsewhere in the wall. Therefore, Chs2 is specific for primary septum formation, whereas Chs3 is responsible for chitin in the ring at bud emergence and in the cell wall. Chs3 is also required for chitin synthesized in the presence of alpha-pheromone or deposited in the cell wall of cdc mutants at nonpermissive temperature, and for chitosan in spore walls. Genetic evidence indicated that a mutant lacking all three chitin synthases was inviable; this was confirmed by constructing a triple mutant rescued by a plasmid carrying a CHS2 gene under control of a GAL1 promoter. Transfer of the mutant from galactose to glucose resulted in cell division arrest followed by cell death. We conclude that some chitin synthesis is essential for viability of yeast cells.

Proteinase B is, indeed, not required for chitin synthetase 1 function in Saccharomyces cerevisiae.

Previous genetic evidence led to the conclusion that proteinase B of yeast was not involved in the function of chitin synthetase 1 (Chs1), based on the demonstration of normal septum formation, cell division and chitin deposition in mutants devoid of the proteinase (Zubenko, G.S., Mitchell, A.P., and Jones, E.W. (1979) Proc. Natl. Acad. Sci. USA 76, 2395-2399). Later, however, it was found that the essential enzyme for septum formation is chitin synthetase 2, whereas Chs1 acts as an auxiliary enzyme, whose absence results in daughter cell lysis under acidic conditions (Cabib, E., Sburlati, A., Bowers, B. and Silverman, S.J. (1989) J. Cell Biol. 108, 1665-1672). By using the lytic behavior as a criterion, we have now found that prb1 strains are not defective in Chs1 function. Certain strains contain a recessive suppressor of lysis which could mask the Chs1 defect. However, appropriate crosses and transformation experiments showed that the prb1 mutants do not harbor the suppressor. It may now be concluded with confidence that proteinase B is not required for chitin synthetase 1 function.

A descriptive analysis of activity structures in high school health education classes.

The extent of use of activity structures in high school health education classes was examined. Twenty health educators from high schools in a large southwestern city agreed to have their classes observed on four separate occasions. Trained individuals observed, coded, and timed various classroom activities using a standardized coding form. Across all 80 observations, most classroom time was spent in Teacher Presentation of Content (23.4%), Seatwork (23.1%), or Media Presentation (15.6%). Little classroom time was used in Student Presentation (.3%) or Behavioral Presentation (.02%). The project goal was to provide baseline observational data into health education classroom activities. From this initial quantitative description of activity structures, correlational and experimental studies can be designed to link these activity patterns with student outcomes in health instruction.

Similar and different domains of chitin synthases 1 and 2 of S. cerevisiae: two isozymes with distinct functions.

Saccharomyces cerevisiae produces two chitin synthases (Chs1 and Chs2) encoded by separate genes. Although these enzymes catalyze the same reaction, Chs2 is essential for septum formation whereas Chs1 has a repair function. To determine if these physiological differences are reflected in the enzyme structures, the CHS2 gene was sequenced and compared to that of CHS1. The predicted amino acid sequence of Chs2 shares substantial similarity with that of Chs1 in the carboxyl two-thirds of the protein. The amino one-third segments differ in predicted isoelectric point by almost 5 pH units. It is suggested that the similar regions are related to common catalytic function. The unrelated regions may be involved in regulation or localization of the respective enzymes. CHS1 and CHS2 are unlinked but may have arisen from the duplication of an ancestral gene.

Chitin synthase 1, an auxiliary enzyme for chitin synthesis in Saccharomyces cerevisiae.

Previously, we showed that chitin synthase 2 (Chs2) is required for septum formation in Saccharomyces cerevisiae, whereas chitin synthase 1 (Chs1) does not appear to be an essential enzyme. However, in strains carrying a disrupted CHS1 gene, frequent lysis of buds is observed. Lysis occurs after nuclear separation and appears to result from damage to the cell wall, as indicated by osmotic stabilization and by a approximately 50-nm orifice at the center of the birth scar. Lysis occurs at a low pH and is prevented by buffering the medium above pH 5. A likely candidate for the lytic system is a previously described chitinase that is probably involved in cell separation. The chitinase has a very acidic pH optimum and a location in the periplasmic space that exposes it to external pH. Accordingly, allosamidin, a specific chitinase inhibitor, substantially reduced the number of lysed cells. Because the presence of Chs1 in the cell abolishes lysis, it is concluded that damage to the cell wall is caused by excessive chitinase activity at acidic pH, which can normally be repaired through chitin synthesis by Chs1. The latter emerges as an auxiliary or emergency enzyme. Other experiments suggest that both Chs1 and Chs2 collaborate in the repair synthesis of chitin, whereas Chs1 cannot substitute for Chs2 in septum formation.

Fungal cell wall synthesis: the construction of a biological structure.

The cell wall is of vital importance for the protection of the fungal cell. It consists of structural components, mostly beta-linked polysaccharides, and of interstitial components, usually glycoproteins. Cell shape is determined by the wall and is attained by localized growth. Regulatory mechanisms that act on chitin and beta(1----3) glucan synthetase indicate how localized biosynthesis can be achieved. Whereas structural polysaccharides appear to be exported by vectorial synthesis through the plasma membrane, glycoproteins are manufactured in an assembly line that takes them through several cellular compartments before exocytosis to the periplasmic space. In this space, the last phase of cell wall construction takes place, i.e. branching of structural polysaccharides and linkage between different components.

Chitin synthase 2 is essential for septum formation and cell division in Saccharomyces cerevisiae.

Previous work led to the puzzling conclusion that chitin synthase 1, the major chitin synthase activity in Saccharomyces cerevisiae, is not required for synthesis of the chitinous primary septum. The mechanism of in vivo synthesis of chitin has now been clarified by cloning the structural gene for the newly found chitin synthase 2, a relatively minor activity in yeast. Disruption of the chitin synthase 2 gene results in the loss of well-defined septa and in growth arrest, establishing that the gene product is essential for both septum formation and cell division.

Differences in nuclear proteins of neurons, astrocytes and C-6 glioma cells.

In an effort to identify cell type specific proteins from brain, we have compared proteins of the cell nucleus from two brain cell types. Using a bulk isolation procedure, we fractionated neurons and astrocytes from adult rat brain. In addition, primary cultures of astrocytes were prepared from one-day old rats. Nuclei from these cells and C-6 glioma cell cultures were isolated and the resulting proteins subjected to two-dimensional gel electrophoresis. Several proteins specific for each cell type were found. While many similarities between bulk brain astrocyte preparations and cultured astrocytes were found, less than pure bulk astrocytes from brain were found to be most similar to those of neurons and not to those from primary cell culture. The nuclear protein profile of cultured astrocytes differed significantly from that of C-6 cells, indicating the utility of two-dimensional gel analysis for detecting major cell type differences in uniform populations of cells.

Changes in nuclear proteins of astrocytes as a result of acute ammonia or ethanol exposure.

We have used an in vitro system to monitor the effects of high levels of ammonia and ethanol on glial cells. Nuclei were isolated and the protein profiles examined by two-dimensional gel electrophoresis. Acute exposure of rat astrocyte cell cultures to ammonia or ethanol resulted in changes in cellular morphology and the level of some nuclear proteins. Glial fibrillary acidic protein (GFAP) levels remained constant under both treatments. Several nuclear proteins were increased specifically. Only one protein was visually detected which was unique to treatment with ammonia or ethanol. This protein (p2a) appeared only in the presence of ammonia. There were no changes in previously observed astrocyte-associated proteins (Silverman et al. Neurochem Int.12, 513-518, 1988). Two proteins appeared de novo upon either treatment with either ammonia or ethanol. These latter proteins had a molecular weight and pI profile similar to the major class of nuclear stress proteins (hsp70). However, results from immunoblot experiments clearly demonstrated that hsp70 was not induced in astroycte cultures following exposure to ammonia or ethanol.

Effects of mycophenolic acid on detection of glial filaments in human and rat astrocytoma cultures.

Expression of glial fibrillary acidic protein (GFAP) was assayed in 11 glioma-derived cell cultures. Treatment of cells with an inhibitor of guanine nucleotide biosynthesis, mycophenolic acid, enhanced detection of GFAP by indirect immunofluorescence microscopy. Quantitation of GFAP and vimentin demonstrated that enhanced fluorescence occurs without an increase in the level of intermediate filament proteins. Immunoblots provided the most sensitive method for monitoring GFAP expression and showed the limitations of using immunofluorescence detection methods. GFAP was detectable in cultures derived from malignant Grade IV astrocytomas and its expression was stable during the course of the study. While mycophenolic acid has been reported to induce differentiation in leukemia cells at low concentration (D.L. Lucas et al., J. Clin. Invest., 72: 1889-1990, 1983), its effect on glioma cultures at concentrations of 100 microM was consistent with a role as an inhibitor of DNA synthesis, and as an effector of altered intermediate filament organization.

Effects of Ty insertions on HIS4 transcription in Saccharomyces cerevisiae.

Insertion of two different Ty elements into the Saccharomyces cerevisiae HIS4 regulatory region eliminates transcription of HIS4. Transcription can be restored by genetic rearrangements involving the Ty element inserted at HIS4. Several deletions, an inversion, a translocation, and a gene conversion are capable of restoring HIS4 transcription. Some of the rearrangements result in new transcriptional initiation sites. One type of revertant of his4-912 results from recombination between the delta elements flanking the Ty element, leaving a solo delta in place of the complete Ty. Strains carrying a Ty912 delta at HIS4 are His- at 23 degrees C. Unlinked suppressors (SPT) lead to suppression of this His- phenotype and increase levels of the normal HIS4 transcript. These suppressor genes affect not only the amount of transcription from the normal HIS4 initiation site, but also that from new initiation sites within Ty sequences adjacent to HIS4.

Regulation of HIS4-lacZ fusions in Saccharomyces cerevisiae.

The beginning of the Saccharomyces cerevisiae HIS4 gene has been fused to the structural gene for Escherichia coli beta-galactosidase. This construction, which contains HIS4 DNA from -732 to +30 relative to the translation initiation codon, has been integrated into the yeast genome at two chromosomal locations, HIS4 and URA3. At both locations, this 762-base-pair stretch of DNA is sufficient for initiating expression of beta-galactosidase activity in S. cerevisiae and confers upon this activity the regulatory response normally found for HIS4.

Bayes' theorem in ophthalmologic computer diagnosis.

Computers are being investigated as diagnostic aids in many fields of medicine. Models employing Bayes' theorem, a statistical formula, commonly are used to supply valuable information on the likelihood of each disease in the differential diagnosis to help the clinician make the diagnosis. However, knowledge of elementary decision analysis is beneficial to help understand the current and potential uses of these models. We discussed Bayes' theorem as an introduction to decision analysis. Moreover, we described a Bayesian model for the differential diagnosis of leukocoria to illustrate the application of computers to ophthalmologic diagnosis.

Bile acids: co-mutagenic activity in the Salmonella-mammalian-microsome mutagenicity test: brief communication.

Of 30 bile acids tested, none was mutagenic in the Salmonella-mammalian-microsome test with indicator strains G46, TA1530, TA1535, TA1536, TA1537, TA1538, TA98, or TA100. However, when lithocholic acid or one of its conjugates was tested with suboptimal amounts of 2-aminoanthracene and phenobarbital-stimulated rat liver homogenate, enhancement and co-mutagenesis were observed if TA1538 was the indicator strain.

Dietary effects on the composition of fecal flora of rats.

A long-term animal feeding experiment was conducted to compare the effect of meat and Wayne laboratory chow diets on the composition of rat fecal flora. Fecal bacteria were enumerated on selective media under both aerobic and anaerobic conditions. Intrarectal administration of N-methyl-N' -nitro-N-nitro-soguanidine (MNNG) affected the count only on the phenylethylalcohol and veillonella-neomycin agars, whereas a slightly higher number of anaerobes appeared in the feces of rats that were treated with MNNG as compared with those obtained in the feces of untreated rats on the meat diet. In the absence of MNNG, feces of meat-fed rats yielded higher bacterial counts on aerobically incubated MacConkey agar, deoxycholate agar, and Pfizer selective enterococcus agar as well as higher numbers of clostridia on anaerobic egg yolk agar than did feces of rats on the Wayne diet. Feces of the group fed the Wayne diet produced more colonies on aerobic mitis-salivarius agar and lactobacillus agar as well as on anaerobically incubated phenylethylalcohol agar, veillonellaneomycin agar, bifidobacteria agar, fusobacterium (Nissui) agar, and kanamycin-vancomycin blood agar. These differences were consistent throughout the 1-year feeding period.