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Cryptococcus neoformans is an opportunistic fungal pathogen that synthesizes and catabolizes inositol. This study demonstrates inositol synthesis from glucose-6-phosphate via inositol-1-phosphate synthase and catabolism to glucuronic acid via inositol oxygenase in this organism. These inositol synthetic and catabolic pathways are regulated in opposition; repressing conditions for one are inducing conditions for the other. An inositol-requiring strain was generated by UV mutagenesis. Without inositol, this mutant strain undergoes 'inositol-less' death, during which time the phosphatidylinositol composition of the membranes decreases without alteration of the proportion of other phospholipids. The mutation on this strain results in no detectable inositol synthetic activity but normal (wild-type) inositol catabolic activity. This inositol-requiring mutant strain reverted at a high frequency. Classical genetic experiments revealed that the majority of the reverting mutations are at second sites. Interestingly, the revertants exhibited unusual morphological phenotypes when deprived of inositol, while provision of inositol restored wild-type morphology. Inositol metabolism is clearly important for growth and development of C. neoformans and may be involved in this organism's mechanism for survival as both a saprophyte in soil and a parasite in humans.

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Trp repressor (25 kDa) is a regulatory protein that controls transcription initiation in the tryptophan biosynthetic operon and at least four other operons in Escherichia coli. An alanine to valine mutation (AV77) in the DNA binding domain is known to increase repressor activity at the trp operator in vivo, but not in vitro. We report here the amide proton exchange rates for the DNA-binding domains of both the wild-type and AV77 proteins. We find that the alanine to valine change stabilizes the flexible DNA-binding domain of the repressor. We present in vivo data showing that, although the AV77 repressor is more inhibitory at the trp operator than the wild-type repressor, it does not have increased activity at the aroH or trpR operator; repression at the aroH operator is, in fact, reduced. Our results suggest that the flexibility exhibited by the wild-type repressor allows a broader range of repressor/DNA interactions, whereas the increased rigidity resulting from the AV77 change limits the repressor's effectiveness at some operators.

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Phosphatidylinositol (PI) synthase (cytidine 5'-diphospho (CDP)-1,2-diacyl-sn-glycerol:myo-inositol 3-phosphatidyltransferase, EC 2.7.8.11) was isolated from the microsomal cell fraction of Candida albicans. The Triton X-100 extracted enzyme was enriched 140-fold by affinity chromatography on CDP-diacylglycerol-Sepharose. The enzyme had a pH optimum at 9.5 in glycine/NaOH buffer. It had an absolute requirement for Mg2+ or Mn2+ and was inhibited by Ca2+ and Zn2+. Maximal activity was at 0.2-0.6 mM-CDP-diacylglycerol, higher concentrations inhibited the enzyme. With 2'-deoxy-CDP-diacylglycerol as the lipid substrate, optimal activity was at 0.7 mM. The K(m) for myo-inositol was determined to be 0.55 mM. The optimal temperature for the PI synthase reaction was 55 degrees C. The C. albicans PI synthase shows differences to the Saccharomyces cerevisiae enzyme, such as activation by bivalent cations, inhibition by nucleotides, temperature optimum and activation energy, but also to the human PI synthase in preference for the lipid substrates, inhibition by nucleoside monophosphates and stabilization by Mn2+ and phospholipids.

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The sequence of the Candida albicans inositol biosynthetic gene, CaINO1, and its flanking regions is determined in this study. The largest open reading frame has a coding sequence of 1560 base pairs, corresponding to a predicted protein of 521 amino acids. Three primary transcriptional start sites are found 64, 57 and 52 base pairs upstream of the ATG translational start site at position 1374. Five stop codons exist in a cluster at the end of the coding region. Within the upstream region TATA and CAAT eukaryotic regulatory sequences are identified along with regions corresponding to a 10 base pair inositol/choline responsive element consensus sequence. Computer analysis of the DNA sequence shows strong homology to the Saccharomyces cerevisiae INO1 gene. A comparison of the deduced amino acid sequence of the C. albicans INO1 gene product, inositol-1-phosphate synthase, with its homolog in S. cerevisiae shows 64% identity and 77% similarity. The differences between the two proteins are most prominent in the N-terminal regions.

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Seven temperature-sensitive cell lysis (cly) mutant strains of Saccharomyces cerevisiae were isolated which lyse at the restrictive temperature on hypotonic but not on osmotically supported medium. The seven mutants fell into four complementation groups, CLY12 to CLY15. The wild-type CLY15 gene was isolated by complementation of the cly15 temperature-sensitive growth defect. Sequence analysis revealed that the complementing DNA fragment encoded a partial PKC1 gene, which has previously been isolated as an S. cerevisiae homolog of mammalian protein kinase C genes (D. E. Levin, F. O. Fields, R. Kunisawa, J. M. Bishop, and J. Thorner, Cell 62:213-224, 1990). Subsequent genetic analysis showed that CLY15 and PKC1 represent identical loci in the yeast genome. A truncated PKC1 gene encoding only the predicted catalytic domain of Pkc1p was able to complement pkc1 mutant strains. Similar to what has been reported recently (D. E. Levin and E. Bartlett-Heubusch, J. Cell Biol. 116:1221-1229, 1992), we observed that cells deleted for the PKC1 gene are viable when grown on osmotically stabilized medium but are osmotically fragile and lyse rapidly after a shift to hypotonic medium. As shown by light and electron microscopic examinations, the delta pkc1 strain exhibits many cells with a strongly elongated bud or chains of incompletely budded cells when grown on solid medium.

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We have previously described a temperature-sensitive pmi40-1 mutant of Saccharomyces cerevisiae which is defective in glycosylation and secretion because of a thermolabile phosphomannose isomerase (PMI) activity. Inactivation of PMI at the restrictive temperature of 37 degrees C prevents synthesis of the GDP-mannose and dolichol-phosphate-mannose required for a number of critical mannosyl transfer reactions and results in cell death. Here, we report the isolation of the PMI40 gene by complementation of the corresponding mutation. The PMI40 gene contains an efficiently spliced intron which differs from the majority of those so far identified in S. cerevisiae in that it is short and the branch-forming structure has an AACTAAC motif replacing the highly conserved consensus TACTAAC. The 48.2-kDa protein predicted to be encoded by PMI40 contains amino acid sequences corresponding to those of internal peptides derived from purified S. cerevisiae PMI. Deletion of the PMI40 coding sequence results in a strain requiring D-mannose for growth. The PMI40 gene is located on chromosome V, and its transcription is increased 12-fold when cells are grown on D-mannose as sole carbon source instead of D-glucose. PMI enzyme activity, however, is not increased in D-mannose-grown cells, and PMI protein levels remain constant, suggesting that the PMI40 gene is subject to additional levels of regulation.

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The Candida albicans inositol biosynthetic gene and its regulation have been studied. The gene, CalNO1, was cloned on a multicopy vector by complementation of a Saccharomyces cerevisae mutant strain. Southern blot analysis established that the cloned DNA was C. albicans genomic DNA in origin; neither rearrangements nor pseudogenes were evident. Blot hybridization analysis using RNA isolated from C. albicans revealed that a single RNA species (1.8 kilobases) was homologous to the cloned DNA fragment. The steady-state levels of these transcripts were shown to be regulated in response to inositol in the growth media. In addition, the steady-state levels of the RNA encoded by the cloned C. albicans DNA present in S. cerevisiae on a plasmid (YRpCalNO1) were regulated in response to exogenously provided inositol. The cloned C. albicans DNA fragment was shown to restore inositol-1-phosphate synthase activity to a S. cerevisiae mutant strain defective in this enzyme. This activity was also shown to be regulated in response to the presence of inositol in the growth media.

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A temperature-sensitive mutant of Saccharomyces cerevisiae was identified which at the restrictive temperature of 37 degrees C is unable to secrete a number of cell wall-associated proteins and thus resembles previously reported sec mutants. In contrast to other sec mutants, however, both the temperature-sensitive growth and the secretion defects can be repaired by the addition of D-mannose to growth media. We show that the mutant possesses a single, apparently recessive mutation which leads to the production of a thermolabile phosphomannose isomerase.

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Phospholipid metabolism in the pathogenic fungus Candida albicans was examined. The phospholipid biosynthetic pathways of C. albicans were elucidated and were shown to be similar to those of Saccharomyces cerevisiae. However, marked differences were seen between these two fungi in the regulation of the pathways in response to exogenously provided precursors inositol and choline. In S. cerevisiae, the biosynthesis of phosphatidylcholine via methylation of phosphatidylethanolamine appears to be regulated in response to inositol and choline; provision of choline alone does not repress the activity of this pathway (G. M. Carman and S. A. Henry, Annu. Rev. Biochem. 58:636-669, 1989). The same pathway in C. albicans responds to the exogenous provision of choline. Possible explanations for the observed differences in regulation are discussed.

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Second-site reversion studies were performed with five missense mutants with defects in the trp repressor of Escherichia coli. These mutants were altered throughout the gene. The same unidirectional mutagen used in the isolation of these mutants, hydroxylamine, was used in reversion studies, to increase the likelihood that the revertants obtained would have second-site changes. Most of the second-site revertants were found to have the same amino acid substitutions detected previously as superrepressor changes. These second-site revertant repressors were more active in vivo than their parental mutant repressors, in the presence or absence of exogenous tryptophan. Apparently superrepressor changes at many locations in this protein can act globally to increase the activity of mutant repressors.

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Interaction of the Escherichia coli trp repressor with the promoter-operator regions of the trp, aroH and trpR operons was studied in vivo and in vitro. The three operators have similar, but non-identical, sequences; each operator is located in a different segment of its respective promoter. In vivo repression of the three operons was measured using single-copy gene fusions to lacZ. The extent of repression varied from 300-fold for the trp operon, to sixfold for the aroH operon and threefold for the trpR operon. To determine whether differential binding of repressor to the three operators was responsible for the differences in repression observed in vivo, three in vitro binding assays were employed. Restriction-site protection, gel retardation and DNase footprinting analyses revealed that repressor binds to the three operators with almost equal affinity. It was also shown in an in vivo competition assay that repressor binds approximately equally well to each of the three operators. It is proposed that the differential regulation observed in vivo may be due to the different relative locations of the three operators within their respective promoters.

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A Saccharomyces cerevisiae mutant (cdg1 mutation) was isolated on the basis of an inositol excretion phenotype and exhibited pleiotropic deficiencies in phospholipid biosynthesis. Genetic analysis of the mutant confirmed that the cdg1 mutation represents a new genetic locus and that a defect in a single gene was responsible for the Cdg1 phenotype. CDP-diacylglycerol synthase activity in mutant haploid cells was 25% of the wild-type derepressed level. Biochemical and immunoblot analyses revealed that the defect in CDP-diacylglycerol synthase activity in the cdg1 mutant was due to a reduced level of the CDP-diacylglycerol synthase Mr-56,000 subunit rather than to an alteration in the enzymological properties of the enzyme. This defect resulted in a reduced rate of CDP-diacylglycerol synthesis, an elevated phosphatidate content, and alterations in overall phospholipid synthesis. Unlike wild-type cells, CDP-diacylglycerol synthase was not regulated in response to water-soluble phospholipid precursors. The cdg1 lesion also caused constitutive expression of inositol-1-phosphate synthase and elevated phosphatidylserine synthase. Phosphatidylinositol synthase was not affected in the cdg1 mutant.

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Availability of the three-dimensional structure of the trp repressor of Escherichia coli and a large group of repressor mutants has permitted the identification and analysis of mutants with substitutions of the amino acid residues that form the tryptophan binding pocket. Mutant aporepressors selected for study were overproduced using a multicopy expression plasmid. Equilibrium dialysis with 14C-tryptophan and purified mutant and wild type aporepressors was employed to determine tryptophan binding constants. The results obtained indicate that replacement of threonine 44 by methionine (TM44) or arginine 84 by histidine (RH84) lowers the affinity for tryptophan approximately two- and four-fold, respectively. Replacement of arginine 54 by histidine (RH84) or glycine 85 by arginine (GR85) results in complete loss of tryptophan binding activity. Purified mutant and wild type aporepressors were used in in vitro heterodimer studies. The trp repressor of E. coli functions as a stable dimer. A large number of trp repressor mutants produces defective repressors that are transdominant to the wild type repressor in vivo. The transdominance presumably results from the formation of inactive or slightly active heterodimers between the mutant and wild type polypeptide subunits. An in vitro assay was developed to detect and measure heterodimer formation. Heterodimer formation was thermally induced, and heterodimers were separated on nondenaturing polyacrylamide gels. Aporepressors readily formed heterodimers upon treatment at 65 degrees C for 3 minutes. Heterodimer formation was significantly retarded by the presence of the corepressor, L-tryptophan. Indole-3-propionic acid, 5-methyl tryptophan, and other analogs of tryptophan, as well as indole, also inhibited heterodimer formation.(ABSTRACT TRUNCATED AT 250 WORDS)

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The mechanism of superrepression by the mutant trp repressor EK49 was examined. This superrepressor has a glutamic acid-to-lysine change at residue 49. The purified EK49 trp repressor was found to have a 10-fold higher affinity than wild type repressor for trp operator DNA. This increased affinity was shown to be due to a decrease in dissociation rate. The binding of trp operator DNA by EK49 trp repressor in the filter binding assay was more sensitive to high salt concentrations than binding by wild type repressor.

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Yeast ino4 mutants are auxotrophic for the phospholipid precursor inositol and have pleiotropic defects in phospholipid synthesis. The mutants are unable to derepress the cytoplasmic enzyme, inositol-1-phosphate synthase and they exhibit reduced synthesis of methylated phospholipids, particularly phosphatidyl-choline. The INO4 gene is believed to encode a positive regulator involved in coordinate control of phospholipid synthesis. In the present study, we report the isolation of two clones containing the INO4 gene. The clones share a region of homology and were mapped to the INO4 locus. Southern blot analysis revealed that the cloned DNA contained both unique and repetitive DNA.

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A filter binding assay was developed that allows measurement of specific binding of trp repressor to operator DNA. The most important feature of this procedure is the concentration and type of salt present in the binding buffer. Using this assay the dissociation constant of the repressor-operator complex was determined to be 2.6 X 10(-9) M, and 1.34 repressor dimers were found to be bound to each operator-containing DNA molecule. These values agree with those obtained by more complex methods. The dissociation constant of the repressor for the corepressor L-tryptophan in the presence of operator DNA was shown to be 2.5 X 10(-5) M. A synthetic 48 bp operator fragment was used to determine the repressor-operator dissociation constant in the presence of tryptophan or tryptophan analogs which have higher or lower affinities for aporepressor. The rate of dissociation of repressor from operator DNA also was determined. Our findings indicate that dissociation is influenced by the concentration of tryptophan or tryptophan analogs and suggest that release of the corepressor may be the first step in dissociation of the repressor-operator complex.

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Phospholipid metabolism in the Saccharomyces cerevisiae opi1 mutant, which excretes inositol and is constitutive for the biosynthetic enzyme inositol-1-phosphate synthase (M. Greenberg, P. Goldwasser, and S. Henry, Mol. Gen. Genet. 186:157-163, 1982), was examined and compared to that of a wild-type strain. In wild-type S. cerevisiae, the phospholipid composition and the relative rates of synthesis of individual phospholipids change in response to the availability of exogenous supplies of soluble phospholipid precursors, particularly inositol. The opi1 mutant, in contrast, displays a relatively invariant phospholipid composition, and its pattern of phospholipid synthesis does not change in response to exogenous phospholipid precursors. Phosphatidylinositol synthase was not found to be regulated in either wild-type or opi1 cells. In wild-type cells, phosphatidylserine synthase and the phospholipid N-methyltransferases are coordinately repressed in response to a combination of inositol and choline. However, in opi1 cells these activities are expressed constitutively. These results suggest that the gene product of the OPI1 locus participates in the coordinate regulation of phospholipid synthesis.

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The Saccharomyces cerevisiae gene, INO1 , encoding the highly regulated enzyme, myo-inositol-1-phosphate synthase [1L-myo-inositol-1-phosphate lyase (isomerizing), EC 5.5.1.4], was isolated by genetic complementation. The cloned sequence was shown to complement two independent IN01 alleles ( ino1 -5 and ino1 -13). One of these mutants ( ino1 -5) fails to make any material that is crossreactive with antibody to the wild-type inositol-1-phosphate synthase. The cloned DNA restored not only inositol prototrophy to this mutant but also its ability to make material crossreactive with anti-inositol-1-phosphate synthase antibody. The sequence on an integrative plasmid was also shown to recombine with the INO1 locus, thereby confirming its genetic identity. The DNA was subcloned and used for Southern blot analysis, revealing that the cloned DNA (5.4 kilobases long) represents a unique sequence in the yeast genome. Inositol-1-phosphate synthase was fully regulated when its gene was located extrachromosomally on the autonomously replicating plasmid. In cells ( ino1 -) containing the cloned INO1 gene on a high-copy-number plasmid, the enzyme was fully repressible. Furthermore, the gene product was not expressed when the plasmid was transferred into a strain containing an ino4 mutation, which also prevents expression of chromosomal copies of INO1 . These results establish that the intact structural gene and associated regulatory components have been isolated and that positioning of the gene in its normal chromosomal location is not required for full regulation of inositol-1-phosphate synthase.

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The structural gene (CHO1) for phosphatidylserine synthase (CDPdiacylglycerol:L-serine O-phosphatidyltransferase, EC 2.7.8.8) was isolated by genetic complementation in Saccharomyces cerevisiae from a bank of yeast genomic DNA on a chimeric plasmid. The cloned DNA (4.0 kilobases long) was shown to represent a unique sequence in the yeast genome. The DNA sequence on an integrative plasmid was shown to recombine into the CHO1 locus, confirming its genetic identity. The cho1 yeast strain transformed with this gene on an autonomously replicating plasmid had significantly increased activity of the regulated membrane-associated enzyme phosphatidylserine synthase. Partial purification of phosphatidylserine synthase from microsomes of this transformed strain confirmed that the membrane-bound enzyme was overproduced 6- to 7-fold as compared with the wild-type strain. The strain also synthesized the product phospholipid, phosphatidylserine, at an increased rate. The transformed strain had altered proportions of a variety of other phospholipids, suggesting that their synthesis is affected by the rate of synthesis of phosphatidylserine in yeast.

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