Deletion of the Candida glabrata ERG3 and ERG11 genes: effect on cell viability, cell growth, sterol composition, and antifungal susceptibility

We have cloned and sequenced the structural genes encoding the delta 5,6 sterol desaturase (ERG3 gene) and the 14 alpha-methyl sterol demethylase (ERG11 gene) from Candida glabrata L5 (leu2). Single and double mutants of these genes were created by gene deletion. The phenotypes of these mutants, including sterol profiles, aerobic viabilities, antifungal susceptibilities, and generation times, were studied. Strain L5D (erg3 delta::LEU2) accumulated mainly ergosta-7,22-dien-3 beta-ol, was aerobically viable, and remained susceptible to antifungal agents but had a slower generation time than its parent strain. L5LUD (LEU2 erg11 delta::URA3) strains required medium supplemented with ergosterol and an anaerobic environment for growth. A spontaneous aerobically viable mutant, L5LUD40R (LEU erg11 delta::URA3), obtained from L5LUD (LEU2 erg11 delta::URA3), was found to accumulate lanosterol and obtusifoliol, was resistant to azole antifungal agents, demonstrated some increase in resistance to amphotericin B, and exhibited a 1.86-fold increase in generation time in comparison with L5 (leu2). The double-deletion mutant L5DUD61 (erg3 delta::LEU2 erg11 delta::URA3) was aerobically viable, produced mainly 14 alpha-methyl fecosterol, and had the same antifungal susceptibility pattern as L5LUD40R (LEU2 erg11 delta::URA3), and its generation time was threefold greater than that of L5 (leu2). Northern (RNA) analysis revealed that the single-deletion mutants had a marked increase in message for the undeleted ERG3 and ERG11 genes. These results indicate that differences in antifungal susceptibilities and the restoration of aerobic viability exist between the C. glabrata ergosterol mutants created in this study and those sterol mutants with similar genetic lesions previously reported for Saccharomyces cerevisiae.     

Site-directed mutagenesis of the phosphocholine-binding site of human C-reactive protein: role of Thr76 and Trp67

We have reported previously that residues Lys57, Arg58, and Trp67 of human C-reactive protein (CRP) contribute to the structure of the phosphocholine (PCh)-binding site. In this study, based on the three-dimensional structures of human CRP and serum amyloid P, we constructed an additional mutant, T76Y, to probe the structural determinants of the PCh-binding site of CRP. Binding properties of four mutant CRPs, K57Q/R58G, W67K, K57Q/R58G/W67K, and T76Y were compared. Wild-type (wt) and all mutant CRPs were purified by affinity chromatography on PCh-, pneumococcal C-polysaccharide (PnC)-, or phosphoethanolamine-conjugated agarose columns. Purified mutant CRPs, K57Q/R58G/W67K and T76Y failed to bind to solid phase, PCh-substituted BSA. They did, however, bind to immobilized PnC, although with substantially decreased avidity compared with wt CRP. W67K, K57Q/R58G/W67K, and T76Y CRP required a 10-fold higher Ca2+ concentration than wt CRP to bind PnC and exhibited decreased avidity for mAb EA4.1, which recognizes a Ca2+-dependent epitope. We conclude that Thr76 is a determinant of the PCh-binding site, probably interacting with the choline group. This conclusion is supported by recent crystallographic data indicating that this residue participates in the formation of a hydrophobic pocket that constitutes the binding site for choline. Trp67, Lys57, and Arg58 do not directly contact PCh, but appear to be required for the proper conformation of the binding site.     

Identification of the orotidine-5'-phosphate decarboxylase gene and development of a transformation system in the yeast Saccharomyces exiguus Yp74L-3

To investigate the uracil biosynthetic pathway of the yeast Saccharomyces exiguus Yp74L-3, uracil auxotrophic mutants were isolated. Using conventional genetic techniques, four mutant genes concerned in uracil biosynthesis were identified and denoted as ura1, ura2, ura3, and ura4. Mutations in the URA3 and URA4 genes were specifically selected with 5-fluoroorotic acid (5-FOA). Vector plasmids containing the URA3 gene and an autonomously replicating sequence (ARS) of S. cerevisiae produced sufficient amounts of Ura+ transformants from the ura4 mutant of S. exiguus. This fact indicates that the S. exiguus URA4 gene encodes orotidine-5'-phosphate decarboxylase (OMP decarboxylase) and demonstrates that vector plasmids for S. cerevisiae are also usable in S. exiguus.     

Chromosomal polymorphism of the yeast Yarrowia lipolytica and related species: electrophoretic karyotyping and hybridization with cloned genes

Significant differences in electrophoretic karyotyping patterns were found among 27 strains of Y. lipolytica. Twenty-one of these strains were classified into four groups of similar karyotypes while six strains showed unique karyotypes. Chromosomal DNAs of different strains were hybridized with cloned genes of Y. lipolytica (URA3, LEU2, ARS18 and ARS68), which revealed four different bands in most strains. We conclude that the haploid chromosome number of Y. lipolytica is at least four, and possibly five or six. Electrophoretic karyotyping and hybridization with cloned genes of Y. lipolytica provided evidence of a large divergence between Y. lipolytica and related species of Saccharomycopsis, Endomycopsella and Endomyces.     

Nucleotide binding sites in wild-type creatine kinase and in W227Y mutant probed by photochemical release of nucleotides and infrared difference spectroscopy

Structural changes induced by nucleotide binding to the wild-type rabbit muscle creatine kinase (CK) and to its W227Y mutant were compared and probed by reaction-induced difference spectroscopy (RIDS). The reaction was induced by the photorelease of nucleotide from the caged nucleotides ADP[Et(PhNO2)] or ATP[Et(PhNO2)], producing the RIDS of CK. The concomitant addition of a saturated concentration of nucleotide and caged nucleotide modified the RIDS of CK, permitting structural changes caused by nucleotide binding in the wild-type creatine kinase to be identified. The W227Y mutant was inactive and its nucleotide binding site was partially impaired as shown by the disappearance or decrease of several nucleotide-sensitive bands in the RIDS of W227Y mutant. The magnitude of the decrease was not the same for each band, suggesting that distinct groups of W227Y mutant were affected differently during nucleotide binding. More precisely, the binding sites for gamma-phosphate and beta-phosphate of the nucleotide were not accessible in W227Y mutant as shown by the absence of the phosphate-sensitive 1666-1667-cm(-1) and 1625-cm(-1) bands in the RIDS of W227Y mutant. However the binding site of other parts of the nucleotide was partially accessible, since the 1638-1639-cm(-1) phosphate-insensitive band did not completely vanish in the RIDS of W227Y mutant. The RIDS of W227Y mutant with ADP[Et(PhNO2)] and creatine lacked the 1613-cm(-1) and 1581-cm(-1) bands, associated with vibrational modes of creatine, suggesting that coupling between the binding sites of the nucleotide and of creatine was altered in W227Y mutant. These results are in accordance with the earlier suggestions that residue W227 in CK is essential for preventing water molecules from penetrating into the active site and for orienting nucleotide in the binding site, by forming stacking interactions between its indole group and purine of the nucleotide and its indole group.     

Interaction between the MEC1-dependent DNA synthesis checkpoint and G1 cyclin function in Saccharomyces cerevisiae

The completion of DNA synthesis in yeast is monitored by a checkpoint that requires MEC1 and RAD53. Here we show that deletion of the Saccharomyces cerevisiae G1 cyclins CLN1 and CLN2 suppressed the essential requirement for MEC1 function. Wild-type levels of CLN1 and CLN2, or overexpression of CLN1, CLN2, or CLB5, but not CLN3, killed mec1 strains. We identified RNR1, which encodes a subunit of ribonucleotide reductase, as a high-copy suppressor of the lethality of mec1 GAL1-CLN1. Northern analysis demonstrated that RNR1 expression is reduced by CLN1 or CLN2 overexpression. Because limiting RNR1 expression would be expected to decrease dNTP pools, CLN1 and CLN2 may cause lethality in mec1 strains by causing initiation of DNA replication with inadequate dNTPs. In contrast to mec1 mutants, MEC1 strains with low dNTPs would be able to delay S phase and thereby remain viable. We propose that the essential function for MEC1 may be the same as its checkpoint function during hydroxyurea treatment, namely, to slow S phase when nucleotides are limiting. In a cln1 cln2 background, a prolonged period of expression of genes turned on at the G1-S border, such as RNR1, has been observed. Thus deletion of CLN1 and CLN2 could function similarly to overexpression of RNR1 in suppressing mec1 lethality.     

Probing the roles of active site residues in phosphatidylinositol-specific phospholipase C from Bacillus cereus by site-directed mutagenesis

The role of amino acid residues located in the active site pocket of phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus[Heinz, D. W., Ryan, M., Bullock, T., & Griffith, O. H. (1995) EMBO J. 14, 3855-3863] was investigated by site-directed mutagenesis, kinetics, and crystal structure analysis. Twelve residues involved in catalysis and substrate binding (His32, Arg69, His82, Gly83, Lys115, Glu117, Arg163, Trp178, Asp180, Asp198, Tyr200, and Asp274) were individually replaced by 1-3 other amino acids, resulting in a total number of 21 mutants. Replacements in the mutants H32A, H32L, R69A, R69E, R69K, H82A, H82L, E117K, R163I, D198A, D198E, D198S, Y200S, and D274S caused essentially complete inactivation of the enzyme. The remaining mutants (G83S, K115E, R163K, W178Y, D180S, Y200F, and D274N) exhibited reduced activities up to 57% when compared with wild-type PI-PLC. Crystal structures determined at a resolution ranging from 2.0 to 2.7 A for six mutants (H32A, H32L, R163K, D198E, D274N, and D274S) showed that significant changes were confined to the site of the respective mutation without perturbation of the rest of the structure. Only in mutant D198E do the side chains of two neighboring arginine residues move across the inositol binding pocket toward the newly introduced glutamic acid. An analysis of these structure-function relationships provides new insight into the catalytic mechanism, and suggests a molecular explanation of some of the substrate stereospecificity and inhibitor binding data available for this enzyme.