The regulatory subunit of protein kinase A promotes hyphal growth and plays an essential role in Yarrowia lipolytica

The gene encoding the regulatory subunit (RKA1) of the cAMP-dependent protein kinase (PKA) of Yarrowia lipolytica was isolated to analyze the role of the PKA pathway in the dimorphic transition of the fungus. The gene encoded a protein of 397 amino acids that exhibits significant homology to fungal PKA regulatory subunits. Attempts to disrupt the gene by double homologous recombination, or the Pop-in Pop-out technique, were unsuccessful. The gene could be mutated only in merodiploids constructed with an autonomous replicating plasmid. Loss of the plasmid occurred with growth under nonselective conditions in the whole population of merodiploids carrying the mutation in the plasmid, but in merodiploids with the mutation at the chromosome, a resistant population prevailed. These data suggest that RKA1 is essential in Y. lipolytica. cAMP addition inhibited the dimorphic transition of the parental strain, but merodiploids carrying several copies of RKA1 were more resistant to cAMP. These results, and the observation that RKA1 was upregulated in mycelial cells, indicate that an active PKA pathway promotes yeast-like growth and opposes mycelial development. This behavior is in contrast to that of Candida albicans, where the PKA pathway favors hyphal growth.     

Regulation of the pattern of protein synthesis in Schizophyllum commune by the incompatibility genes

The pattern of protein synthesis in various coisogenic mycelial types of Schizophyllum commune, viz. monokaryon, dikaryon, and homokaryons carrying primary mutations in the A and the B factors, was studied by two-dimensional gel electrophoresis. After pulse-labeling with ³⁵S-methionine, approximately 650 of 710 proteins analyzed were common to all mycelial types. Coisogenic monokaryons differed by only 2%, whereas the largest difference was found between these monokaryons and the dikaryon derived from them (6.6 and 7.7%). The majority of these differences fell into two about equally sized categories, i.e., proteins which were either specifically absent (“switched-off” proteins) or present (“switched-on” proteins) in the dikaryon. “Switched-on” proteins were on the average larger and slightly more acidic than “switched-off” proteins. The double factor mutant which best mimicked the dikaryon in morphology also best resembled the dikaryon in types of proteins synthesized. Unexpected, however, was the large overlap in proteins apparently controlled by each of the two incompatibility factors individually, despite the distinct morphological sequences directed by each of them.     

Transformation of Bacillus subtilis competent cells: Identification of a protein involved in recombination

Summary With the use of two-dimensional gel electrophoresis, the proteins present in a transformation-proficient B. subtilis strain were compared with those present in an isogenic, recombination-deficient strain carrying the recE4 mutation. One protein (molecular weight 45 kD, iso-electric point 5.4) was found to be virtually absent in the recE4 strain. This 45 kD protein is a prominent protein predominantly present in the competent fraction of a competent culture. The synthesis of the protein is substantially stimulated by irradiation with ultraviolet light or treatment with mitomycin C and, to a lesser extent, by treatment with nalidixic acid. Since the protein is also observed in a strain cured for SP and carrying non-inducible PBS X, it is unlikely that this protein is a gene product specified by one of these prophages usually present in B. subtilis strain 168. Based on these results we conclude that the 45 kD protein is involved in recombination in B. subtilis.     

The Saccharomyces cerevisiae PUT3 activator protein associates with proline-specific upstream activation sequences.

The PUT1 and PUT2 genes encoding the enzymes of the proline utilization pathway of Saccharomyces cerevisiae are induced by proline and activated by the product of the PUT3 gene. Two upstream activation sequences (UASs) in the PUT1 promoter were identified by homology to the PUT2 UAS. Deletion analysis of the two PUT1 UASs showed that they were functionally independent and additive in producing maximal levels of gene expression. The consensus PUT UAS is a 21-base-pair partially palindromic sequence required in vivo for induction of both genes. The results of a gel mobility shift assay demonstrated that the proline-specific UAS is the binding site of a protein factor. In vitro complex formation was observed in crude extracts of yeast strains carrying either a single genomic copy of the PUT3 gene or the cloned PUT3 gene on a 2 microns plasmid, and the binding was dosage dependent. DNA-binding activity was not observed in extracts of strains carrying either a put3 mutation that caused a noninducible (Put-) phenotype or a deletion of the gene. Wild-type levels of complex formation were observed in an extract of a strain carrying an allele of PUT3 that resulted in a constitutive (Put+) phenotype. Extracts from a strain carrying a PUT3-lacZ gene fusion formed two complexes of slower mobility than the wild-type complex. We conclude that the PUT3 product is either a DNA-binding protein or part of a DNA-binding complex that recognizes the UASs of both PUT1 and PUT2. Binding was observed in extracts of a strain grown in the presence or absence of proline, demonstrating the constitutive nature of the DNA-protein interaction.     

Biochemistry of Sexual Morphogenesis in Schizophyllum commune: Effect of Mutations Affecting the Incompatibility System on Cell-Wall Metabolism

A number of homokaryons of Schizophyllum commune, which carry various mutations affecting the incompatibility system, and a wild-type homokaryon were grown and examined for differences in the net synthesis of the cell-wall polysaccharides S-glucan, R-glucan, and chitin and in the activity of an enzyme hydrolyzing R-glucan (R-glucanase) in mycelial extracts and culture media. Only slight differences were observed for the accumulation of S-glucan and chitin. In comparison with the wild-type homokaryon, a very high S-glucan/R-glucan ratio was found in a primary B-factor mutant strain. Essentially, wild-type S-glucan/R-glucan ratios were restored in two strains in which additional mutations restored normal morphology: a strain carrying a secondary B-factor mutation and a strain carrying a modifier mutation in addition to the primary B-factor mutation. The S-glucan/R-glucan ratios in three A-factor mutants were intermediate between those of the wild-type homokaryon and the primary B-factor mutant. In young, growing cultures of the various homokaryons, except for the A-factor mutants, a correlation was found between the S-glucan/R-glucan ratios in the cell wall and the activities of R-glucanase in mycelial extracts. A certain specificity of the effect of the studied mutations on enzyme activities was indicated by the fact that, in young cultures, changes in R-glucanase activities were not paralleled by similar changes in the activities of laminarinase and maltase. The results can be correlated with particular morphological features of the homokaryons and, together with earlier results obtained with heterokaryons, indicate the activity of R-glucanase as an integral component of sexual morphogenesis regulated by the incompatibility factors.     

Cell wall composition of the yeast and mycelial forms of Yarrowia lipolytica

The cell walls of the yeast and mycelial forms of Yarrowia lipolytica were isolated and purified. Electron microscopy studies showed no differences between both types of cell walls. Chemical analysis revealed that the yeast cell wall contained 70% neutral carbohydrate, 7% amino sugars, 15% protein, 5% lipids and 0.8% phosphorus. Mycelial cell walls contained 70% carbohydrate, 14% aminosugars, 6% protein, 5% lipids and 0.6% phosphorus. Three polysaccharides: -glucan, mannan and chitin were detected. Proteins were solubilized from both cell wall fractions and separated by polyacrylamide gel electrophoresis. About 50 protein bands were detected, four of them corresponding to glycoproteins. The cell walls of the yeast and mycelial forms of Y. lipolytica were qualitatively similar and only quantitative differences were found.Abbreviations GlcNAc N-acetylglucosamine - FITC-WGA fluorescein isothiocyanate-wheat germ agglutinin - PAS periodic acid Schiff     

The kinetics of mycelial growth.

Based on the assumption that mycelial growth follows the logistic growth law, formulae have been developed to express the growth of fungal colonies under a variety of geometric constraints. Analysis was done of Deppe's (1973) results on surface colony growth, where the mass of the colony grew exponentially during most colonial growth, and of Trinci's (1970) results on submerged "pellet" growth, where the mass of the colony increased as the cube of time during most colony growth. In both cases, the linear dimensions of the colony were increasing linearly while the mass was changing in these quantitatively different manners. It is concluded that these disparate growth behaviours result from different habits of growth; in two-dimensional colony growth a new region of space if invaded by an amount of mycelium small in proportion to the final "carrying capacity" of the region, and in three-dimensional colony growth a region is invaded with an amount of mycelium almost equal to the region's final limiting mycelial mass. Thus, the types of growth law for colony mass which are applicable for a particular organism in a particular physical environment depend critically on the degree to which the invading hyphae initially occupy the space.     

Studies on Aspergillus Fumigatus; Hydrolysis of Polyamino Acids by Mycelial Filtrate and Chromatographic Fractionation of the Filtrate

Mycelial filtrates from Aspergillus fumigatus (AF), shown to possess haemolytic, toxic, casein precipitating, and protein hydrolyzing activity, hydrolyzed poly-L-lysine and poly-L-glutamine in the pH range 4.6—5.3. Incipient activity against poly-L-lysin was observed at pH 9. Owing to spontaneous hydrolysis of the polyamino acid at pH > 10, no activity optimum could be traced. Gel filtration of mycelial filtrate on Sephadex G-75 or G-100 columns offered no definite information whether the protein hydrolyzing activity, using haemoglobin as substrate, at the optimum pH values, 2.9, 4.6 and 10, shows the activity of a single enzyme with more than 1 pH optimum or of more than 1 enzyme active at different pH values. Certain results of the investigations seem to indicate that the protein hydrolyzing activity at pH 2.9 was not caused by enzymes identical with the enzyme (s) causing the protein hydrolyzing activity at pH 4.6 or pH 10. Casein precipitating and protein hydrolyzing activity occurred, following gel filtration on a Sephadex G-100 column, in identical fractions whereas neither haemolysin nor toxin could be found in samples of 0.5 ml fraction solution from any of the fractions after filtration on Sephadex G-75 or G-100 columns. By using antiserum to a crude filtrate from a homologous AF strain it could be shown, applying immuno-electrophoresis, that dialyzed mycelial filtrate contained 8 precipitating antigens whereas proteinase purified by gel filtration and displaying protein hydrolyzing activity at pH 2.9, pH 4.6 and pH 10 contained 4 such antigens.     

Targeted gene mutation in Phytophthora spp

The genus Phytophthora belongs to the oomycetes and is composed of plant pathogens. Currently, there are no strategies to mutate specific genes for members of this genus. Whole genome sequences are available or being prepared for Phytophthora sojae, P. ramorum, P. infestans, and P. capsici and the development of molecular biological techniques for functional genomics is encouraged. This article describes the adaptation of the reverse-genetic strategy of targeting induced local lesions in genomes (TILLING) to isolate gene-specific mutants in Phytophthora spp. A genomic library of 2,400 ethylnitrosourea (ENU) mutants of P. sojae was created and screened for induced point mutations in the genes encoding a necrosisinducing protein (PsojNIP) and a Phytophthora-specific phospholipase D (PsPXTM-PLD). Mutations were detected in single individuals and included silent, missense, and nonsense changes. Homozygous mutant isolates carrying a potentially deleterious missense mutation in PsojNIP and a premature stop codon in PsPXTM-PLD were identified. No phenotypic effect has yet been found for the homozygous mutant of PsojNIP. For those of PsPXTM-PLD, a reduction in growth rate and an appressed mycelial growth was observed. This demonstrates the feasibility of target-selected gene disruption for Phytophthora spp. and adds an important tool for functional genomic investigation.     

Controlled mycelial growth for kojic acid production using Ca-alginate-immobilized fungal cells

Summary Conidia of Aspergillus oryzae were immobilized in Ca-alginate beads and then incubated in a nutrient medium to yield an immobilized biocatalyst producing kojic acid. The immobilized cell cultures produced kojic acid linearly during cultivation. Regardless of the size of the immobilized particles, there existed an optimal nitrogen concentration for the maximum production rate of kojic acid, at which smaller bead sizes resulted in a higher production rate. When the growth of mycelia were confined within the bead surface and segregated from each other by gel material, they produced kojic acid with maximal catalytic activity and exhibited the highest conversion yield of glucose. The extent of mycelial segregation was especially higher in cultures of smaller bead particles, and the depth of mycelial growth was 150 to 250 m from the gel bead surface in all cultures of different nitrogen concentrations and bead sizes. Therefore, for the maximum expression of catalytic activities of immobilized mycelial cultures, it was found very critical to optimally control the mycelial distribution in gel beads by the culture conditions affecting mycelial growth.     

Microsequence analysis of electroblotted proteins. I. Comparison of electroblotting recoveries using different types of PVDF membranes.

The most effective protein purification method of low picomole amounts for sequence analysis involves polyacrylamide gel electrophoresis followed by electroblotting to polyvinylidene difluoride (PVDF) membranes. Since a critical factor in this procedure is the protein recovery at the blotting step, different types of PVDF membranes were systematically evaluated for their ability to bind proteins during electrotransfer. Differences in electroblotting recoveries occurred between types of PVDF membranes for some proteins. Some variability persisted even when optimized electroblotting procedures were used which reduce the sodium dodecyl sulfate (SDS) concentration in the gel and improve protein-PVDF binding. The membranes which were evaluated could be grouped as either "high retention" membranes (ProBlott, Trans-Blot, and Immobilon-PSQ) or "low retention" membranes (Immobilon-P and Westran). The high retention membranes showed higher protein recoveries under most conditions tested, especially for small proteins or peptides. These high retention membranes were also less sensitive to the exact electroblotting conditions, especially those factors which affect the amount of SDS present during either electrotransfer or direct adsorption from protein solutions. High retention PVDF membranes are therefore preferred in most cases for optimal protein or peptide recovery prior to direct sequence analysis. In contrast, low retention membranes are preferred for procedures where subsequent extraction of the proteins from the membranes is required. Even under identical conditions, substantial protein-to-protein variation for both adsorption and subsequent extraction is routinely observed for both groups of membranes, indicating that the nature of protein-PVDF interactions is more complex than simple hydrophobic interactions.     

Chitinase-like proteins with antifungal activity from emperor banana fruits

Two 30-kDa proteins with N-terminal sequence homology to chitinases have been isolated from fruits of the emperor banana by using a protocol that involved (NH(4))(2)SO(4) precipitation, affinity chromatography on Affi-gel blue gel, ion exchange chromatography by fast protein liquid chromatography (FPLC) on Mono S and gel filtration by FPLC on Superdex 75. The proteins were adsorbed on Affi-gel blue gel and Mono S. They both inhibited mycelial growth in Fusarium oxysporum but not in Mycosphaerella arachidicola. The chitinase-like protein more strongly bound on Mono S was obtained with a slightly lower yield and exhibited a higher antifungal potency toward F. oxysporum when compared with the less strongly bound chitinase-like protein.     

Preliminary characterization of the material released to the culture medium byCandida albicans yeast and mycelial cells

Culture filtrate concentrates were obtained fromCandida albicans yeast and mycelial cells grown in the presence of¹⁴C-protein hydrolysate for radioactive labeling of cellular polypeptides. Both growth forms released to the medium minor but significant amounts of proteinaceous materials. The analysis of culture filtrate concentrates by means of SDS-polyacrylamide gel electrophoresis and fluorography revealed a similar and complex electrophoretic pattern, though some qualitative and quantitative differences between samples obtained from yeast and mycelial cells were observed. Materials released, mostly composed of mannoproteins as shown by their affinity towards concanavalin A, presented (i) cross-reactivity (by Western immunoblotting and ELISA) against polyclonal antisera to genuine cell wall components (among them the 58-kilodalton fibrinogen-binding mannoprotein) and (ii) high affinity for polystyrene-latex microbeads. Results presented suggest a possible common identity for the molecules shed to the medium and the cell wall protein and mannoprotein constituents.Abbreviations SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electroporesis - PAS periodic acid/Schiff method     

A proteomic analysis of changes in prothrombin and plasma proteins associated with the G20210A mutation

The G-->A mutation at position 20210 of the prothrombin gene, localized in the 3'-polyadenylation untranslated region of the mRNA, is a recognized genetic risk factor for venous thromboembolism. The mechanism by which this base change confers an increased risk of thrombosis compared to noncarriers is undefined. Studies on the mRNA suggest enhanced cleavage site recognition and a change in the location of the 3'-cleavage/polyadenylation reaction, but no defined model has been proposed. The present study, based on proteomic investigation by two-dimensional gel electrophoresis and electrospray ionization (ESI) tandem mass spectrometry (MS/MS) protein identification, suggests that the G20210A mutation is associated with increased glycosylation of prothrombin, which confers greater stability to the protein. Additionally, proteomic investigation of pooled plasma showed that expression levels of six spots, three of them identified by ESI MS/MS, were altered in subjects carrying the mutation, suggesting a possible cooperative effect in the thrombotic risk increment induced by the mutation.     

The “8-kD” Cytoplasmic Dynein Light Chain Is Required for Nuclear Migration and for Dynein Heavy Chain Localization in Aspergillus nidulans

The heavy chain of cytoplasmic dynein is required for nuclear migration in Aspergillus nidulans and other fungi. Here we report on a new gene required for nuclear migration, nudG, which encodes a homologue of the “8-kD” cytoplasmic dynein light chain (CDLC). We demonstrate that the temperature sensitive nudG8 mutation inhibits nuclear migration and growth at restrictive temperature. This mutation also inhibits asexual and sexual sporulation, decreases the intracellular concentration of the nudG CDLC protein and causes the cytoplasmic dynein heavy chain to be absent from the mycelial tip, where it is normally located in wild-type mycelia. Coimmunoprecipitation experiments with antibodies against the cytoplasmic dynein heavy chain (CDHC) and the nudG CDLC demonstrated that some fraction of the cytoplasmic dynein light chain is in a protein complex with the CDHC. Sucrose gradient sedimentation analysis, however, showed that not all of the NUDG protein is complexed with the heavy chain. A double mutant carrying a cytoplasmic dynein heavy chain deletion plus a temperature-sensitive nudG mutation grew no more slowly at restrictive temperature than a strain with only the CDHC deletion. This result demonstrates that the effect of the nudG mutation on nuclear migration and growth is mediated through an interaction with the CDHC rather than with some other molecule (e.g., myosin-V) with which the 8-kD CDLC might theoretically interact.     

Analysis of helicase gene mutations in Japanese Werner’s syndrome patients

The profile of helicase gene mutations was studied in 89 Japanese Werner’s syndrome (WRN) patients by examining the previously described mutations 1– 4 as well as a new mutation found during this study, designated mutation 5. Of 178 chromosomes (89 patients), 89 chromosomes (50%) had mutation 4, 11 (6.2%) chromosomes had mutation 1, and two chromosomes (1.1%) contained mutation 5. Mutations 2 and 3 were not observed in this patient population. The remaining 76 (42.7%) chromosomes had none of these mutations. A significant fraction of all patients (22 total patients, 24.7%) appear to be compound heterozygotes, including those carrying mutations of both types 1 and 4. The genotype analysis of the markers surrounding the WRN helicase gene strongly suggests that most of the chromosomes carrying either mutation 1 or 4 were derived from two single founders. Received: 25 July 1996 / Revised: 20 September 1996     

Physiology of a colony of<Emphasis Type="Italic"> Pleurotus pulmonarius</Emphasis> grown on medium overlaid with a Cellophane membrane

The physiology of a colony of Pleurotus pulmonarius grown on potato dextrose agar overlaid with a Cellophane membrane (PDA-WC) was studied. On PDA-WC, the colony presented higher biomass density and productivity of fruit body formation (4.84±0.23 mg mycelial biomass/cm² and 23.7±3.12 g ml^–1 h^–1, respectively) than that which developed on potato dextrose agar without Cellophane (PDA-OC; 0.26±0.01 mg mycelial biomass/cm² and 10.8±1.57 g ml^–1 h^–1, respectively). In cultures developed on PDA-WC, intracellular laccases and -1,3-glucanases activities were lower [12±0.9 arbitrary units (AU)/g mycelial biomass and 1.33±0.1 international units (IU)/g mycelial biomass, respectively] than those observed on PDA-OC (20.65±1.0 AU/g mycelial biomass and 3.67±0.2 IU/g mycelial biomass, respectively). In cultures developed on PDA-WC, intracellular protein and glycogen concentrations were lower (1.9±0.9 and 117±3.5 mg/g mycelial biomass, respectively) than those observed on PDA-OC (14.3±1.1 and 347±2.9 mg/g mycelial biomass, respectively). The radial growth rate and the content of glucans in the cell wall were not significantly different between cultures developed on PDA-WC and PDA-OC. These results show that the use of the Cellophane as a tool to study in vitro fungal physiology might affect the interpretation of experimental results, since the physiology under otherwise similar conditions was different on medium with and without Cellophane.     

A Plasma Membrane Mutation acrA in Escherichia coli

Protein analysis and electron microscopic observation of thefreeze-fractured plane of the plasma membrane were performedwith an acriflavine-sensitive mutant carrying mutation acrA(at min 10) and with the wild type (acrA⁺) strain of Escherichiacoli K-12. The acrA mutant membrane was deficient (or much lower)in one protein when analyzed by the polyacrylamide gel electrophoresistechnique. (Received May 7, 1981; Accepted July 28, 1981)     

鼠伤寒沙门氏菌嘌呤生物合成调控研究：Ⅸ.PURbox的突变分析2

为研究１６ｂｐ ＰＵＲｂｏｓ中８个完全保守的碱基中的２个碱基与ｐｕｒＲ＾＋阻遏蛋白结合中的功能,对它们分别作了定点突变,使其分别从Ｃ,Ｇ突变为Ｇ,Ａ。凝胶阻滞实验结果表明,含上述保守碱基突变的ＰＵＲ ｂｏｘ均不能与ｐｕｒＲ＾＋阻遏蛋白结合。证明这２个保守碱基对维持ＰＵＲｂｏｘ的功能是必须的,其中任一改变都导致ＰＵＲｂｏｘ功能的丧失。     

Macroporous polyacrylamide monolithic gels with immobilized metal affinity ligands: the effect of porous structure and ligand coupling chemistry on protein binding

Macroporous polyacrylamide gels (MPAAG) with iminodiacetic acid (IDA) functionality were prepared by (i) chemical modification of polyacrylamide gel, (ii) co-polymerization of acrylamide with allyl glycidyl ether (AGE) and N,N'metylene-bis(acrylamide) (MBAAm) followed by coupling IDA ligand or (iii) by copolymerization of acrylamide and MBAAm with functional monomer carrying IDA-functionality (1-(N,N-bis(carboxymethyl)amino-3-allylglycerol). Screening for optimized conditions for the production of the MPAAG with required porous properties was performed in a 96-well chromatographic format that allowed parallel production and analysis of the MPAAG prepared from reaction mixtures with different compositions. Scanning electron microscopy of the fabricated MPAAG revealed two different types of the porous structures: monomodal macroporous structure with large interconnected pores separated by dense non-porous pore walls in case of plain gels or gels produced via copolymerization with AGE. The other type of the MPAAG (gel produced via co-polymerization with functional monomer carrying IDA-functionality) had bimodal pore structure with large interconnected pores separated by the pore walls pierced through with micropores. The effect of different modifications of MPAAG monoliths and of porous structure of the MPAAG (monomodal and bimodal porous structure) on protein binding has been evaluated.