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PubMed1987

Spore activation by acetate, propionate and heat in Phycomyces mutants.

Exposure to heat, acetate, or propionate activates the spores of the fungus Phycomyces blakesleeanus and allows them to germinate. Using counterselection with the antibiotic N-glycosyl-polyfungin, seven mutants were isolated on the basis of decreased spore activation by propionate. The nine mutants showed decreased activation by both chemicals and by heat, increased heat lethality, and altered patterns of trehalase activation. These and other observations indicate that spore activation by the three agents and spore death by heat are mediated by the same cellular component(s), which is probably involved in the regulation of cyclic AMP concentration.

Acetates↗
PubMed1983

Genetic analysis of hypertropic mutants of Phycomyces.

A new class of Phycomyces behavioral mutants with enhanced tropic responses has been analyzed genetically to determine the number of genes involved and the nature of their expression. These hypertropic mutants carry pleiotropic nuclear mutations. Besides their effects on sensory behavior, they also affect morphology and meiotic processes. Behavioral analyses of heterokaryons containing hypertropic and wild-type nuclei in varying proportions show that the hypertropic mutations in strains L82, L84, L86, and L88 are strongly dominant. Conversely, the hypertropic mutations carried by the strains L83, L85, and L87 are strongly recessive. We performed recombination analyses between hypertropic mutants and mutants with diminished phototropism, affected in the seven genes madA to madG. We found no evidence of linkage between the hypertropic mutations and any of these mad mutations. From crosses, we isolated double mutants carrying hypertropic mutations together with madC (night blind) and madG (stiff) mutations. The behavioral phenotypes of the double mutants are intermediate between those of the parentals. Complementation analyses show that the three recessive hypertropic mutations affect the same gene, which we call madH. The expression of the recessive hypertropic allele becomes dominant in heterokaryons carrying madC and madH nuclei; the madC gene has been implicated separately with the photoreceptor at the input to the sensory pathway, while the madH gene is associated with the growth control output. This result suggests the physical interaction of both gene products, madH and madC, in a molecular complex for the photosensory transduction chain.

Fungi↗
PubMed1988

Expression of Tn5-derived kanamycin resistance in the fungus Phycomyces blakesleeanus.

A plasmid, carrying the Tn5 gene for kanamycin resistance lacking its own promoter, has successfully been used in the selection of DNA sequences of the fungus Phycomyces blakesleeanus having promoter activity in Escherichia coli. Many of these sequences were also effective in promoting resistance to kanamycin when the corresponding chimeric plasmids were introduced in the fungus via spheroplast transformation. The selected phenotype was easily propagated through vegetative spores and behaved as a stable character since it was not appreciably lost in the absence of selection.

DNA Transposable Elements↗
PubMed1992

Heterologous transformation of Mucor circinelloides with the Phycomyces blakesleeanus leu1 gene.

The leu1 gene of Phycomyces blakesleeanus was isolated within a HindIII-HindIII genomic DNA fragment by heterologous hybridization screening of a cosmid library, making use of the Mucor circinelloides leuA gene as a probe. The complete nucleotide sequence of this fragment reveals a single 2070 bp ORF with no introns, which presents at least 68% homology with that of the leuA gene. The P. blakesleeanus leu1 gene has also been expressed in the M. circinelloides mutant R7B (leu-), which was used to isolate the leuA gene by complementation. The homology with other known sequences shows that the leu1 gene encodes the P. blakesleeanus alpha-IPM (isopropylmalate) isomerase.

Amino Acid Sequence↗
PubMed1987

A genetic map of Phycomyces blakesleeanus.

Complementation tests among Phycomyces auxotrophic strains revealed the existence of four genes with mutants requiring riboflavin, three genes with purine auxotrophs, two with nicotinic acid auxotrophs, and two with lysine auxotrophs. A total of 134 sexual crosses between strains carrying mutations affecting phototropism (madA-madE), carotenoid biosynthesis (carA), auxotrophy (ribA-ribD, purA-purC, lysA and lysB, nicA and nicB, and leuA) and resistance to 5-fluorouracil (furA and furB) were studied; mating type (sex) was also included as a marker. The results from random spore analysis, tetrad analysis, and gene-centromere distances shows that these markers are distributed into 11 linkage groups.

Chromosome Mapping↗
PubMed1986

System analysis of Phycomyces light-growth response: single mutants.

The white-noise method of system identification has been applied to the transient light-growth response of a set of seven mutants of Phycomyces with abnormal phototropism, affected in genes madA to madG. The Wiener kernels, which represent the input-output relation of the light-growth response, have been evaluated for each of these mutants and the wild-type strain at a log-mean blue-light intensity of 0.1 W m-2. Additional experiments were done at 3 X 10(-4) and 10 W m-2 on the madA strain C21 and wild-type. In the normal intensity range (0.1 W m-2) the madA mutant behaves similarly to wild-type, but, at high intensity, the madA response is about twice as strong as that of wild-type. Except for C21 (madA), the first-order kernels of all mutants were smaller than the wild-type kernel. The first-order kernels for C111 (madB) and L15 (madC) show a prolonged time course, and C111 has a longer latency. The kernels for C110 (madE), C316 (madF), and C307 (madG) have a shallow and extended negative phase. For C68 (madD), the latency and time course are shorter than in the wild-type. These features are also reflected in the parameters estimated from fits of the analytical model introduced in the previous paper to the experimental transfer functions (Fourier transforms of the kernels). The kernel for L15 (madC) is described better by a model that lacks one of the two second-order low-pass filters, because its response kinetics are dynamically of lower order.

Light↗
PubMed1986

System analysis of Phycomyces light-growth response: double mutants.

The light-growth response of Phycomyces has been studied with Gaussian white-noise test stimuli for a set of 21 double mutants affected in all pairwise combinations of genes madA to madG; these genes are associated with phototropism, the light-growth response, and other behaviors. The input-output relations of the light-growth responses of these mutants are represented by Wiener kernels in the time domain and transfer functions in the frequency domain. The results have been analyzed comparatively with those in the preceding papers on wild-type and single mutant strains. Two of the double night-blind mutants (combinations AB and BC) have especially weak, but still detectable, responses. To evaluate possible dynamic interactions among the seven mad gene products, each double-mutant transfer function was analyzed jointly with those of the parental single mutants and wild-type. Specifically, a hypothesis of dynamic independence was rejected at the 5% significance level for the following combinations: AD, AE, AG, BC, BD, BE, BF, BG, CD, CE, CF, DE, DG, and EF. A formal pictorial scheme summarizes the dynamic interactions among the mad gene products, according to this test. The high degree of interactions between the "input" gene products (A, B, and C) and the "output" gene products (D, E, F, and G) suggest that most or all of the sensory transduction pathway for the light-growth response (and phototropism) is contained in a multimolecular complex.

Light↗
PubMed1993

Isolation, characterization and mapping of pyrimidine auxotrophs of Phycomyces blakesleeanus.

A total of seven pyrimidine auxotrophs of Phycomyces were isolated from among 5-fluoroorotate acid (5-FOA)-resistant mutants. They were classified by complementation into two groups. A representative mutant strain belonging to one group was deficient in orotate phosphoribosyl transferase (OPRTase; EC 2.4.2.10) activity; the mutant strain belonging to the second group was deficient in orotidine-5'-monophosphate decarboxylase (OMPdecase; EC 4.1.1.23). These mutants are defective in the genes pyrF and pyrG respectively. The results from random spore analysis, tetrad analysis, and gene-centromere distances showed that these two markers are located in linkage group VI, with pyrG being a proximal marker and pyrF a distal one.

Cell-Free System↗
PubMed1986

A method for the selection of mutants of Phycomyces blakesleeanus defective in germination.

A simple method for the physical separation of dormant and germinated cells of Phycomyces blakesleeanus has been designed. Separation was attained by isopycnic centrifugation in a preformed discontinuous density gradient of Urografin. In this gradient, dormant spores and active cells at two different stages of germination were separated in three homogeneous and clearly distinct bands. The method has been used to follow the kinetics of the variation of cellular density during germination of wild type spores and to select for mutants temperature-sensitive for heat-shock induced germination. Sixty-nine mutants have been isolated by various procedures which include a separation step in the described Urografin gradient.

Centrifugation, Density Gradient↗
PubMed1986

Characterization of Phycomyces blakesleeanus mutants temperature-sensitive for heat-shock induced germination.

Sixty-nine mutants of the fungus Phycomyces blakesleeanus, temperature-sensitive for heat-shock induced germination, have been characterized. All of them show a low viability at 26 degrees C and normal viability at 16 degrees C. Eleven mutants recover the wild type phenotype if yeast extract is added to the minimal medium; the mutant phenotype of eight of these mutants is also suppressed by the addition of putrescine or other polyamines. The majority of the mutants are affected very early in germination. Spontaneous, heat-shock and acetate induced germination are not equally impaired by some of the mutations, so specific and independent steps seem to be involved in part of the activation mechanism of germination.

Genes, Fungal↗
PubMed1977

Cell wall carbohydrates in Phycomyces blakeslesanus Burgeff.

The carbohydrate composition of the cell walls from spores, mycelium and sporangiophores of Phycomyces blakesleeanus was analyzed. Spore wall polysaccharides contained over 50% glucose, about 20% uronic acids, 10% mannose and 10% amino-sugars. During the growth of the hyphae amino-sugars became the main carbohydrate (45%); uronic acids contributed some 25%, glucose and fructose 10% and galactose nearly 6%. Sporangiophores contained almost 90% aminosurgars and some 6% uronic acids. Traces of rhamnose were found in all wall preparations. A similar picture emerged from studies on the incorporation of [U-14C]-glucose into wall materials. Furthermore we looked for a GDP-fucose synthesizing system and found an increasing activity during early germination. This rise in activity was inhibited by cycloheximide but only by 5-fluorouracil.

Amino Sugars↗
PubMed1990

Chitin synthetase activity in a developmental mutant of Phycomyces blakesleeanus.

Chitin synthetase activity was analyzed in vitro and in vivo in two morphogenetic stages, namely, dormant spore cells and germlings of the wild type strain and the developmental mutant S356 of Phycomyces blakesleeanus. In vitro experiments showed a much higher specific activity in dormant spores of the mutant strain than in those of the wild-type. This difference was restricted to the dormant spore phase since germlings exhibited comparable levels of activity to those detected in the wild-type strain. Although no correlation was observed between chitin synthesis in vitro and in vivo in mutant spores, germination of these cells was accompanied by an earlier expression of chitin synthetase in vivo. Germination of mutant spores in liquid medium produced morphologically aberrant germlings. Contrary to the extended mycelial growth of the wild-type strain in solid medium, the mutant grew with a typical colonial morphology. Results are discussed in relation to the possible basis of the mutant phenotype.

Chitin Synthase↗
PubMed1976

Effect of 5-fluorouracil and cycloheximide on the early development of Phycomyces blakesleeanus spores and the activity of N-acetylglucosamine synthesizing enzymes.

The development of germinating Phycomyces spores was not inhibited by 5-fluorouracil (1mM) until the emergence of the germination tube. Fluorouracil was incorporated into RNA as efficiently as uracil; it did not inhibit the synthesis of proteins and the increase in respiratory activity during early development. Cycloheximide inhibited development as well as the increase in respiration and protein synthesis. This suggested that protein synthesis or some other cycloheximide dependent process, but no mRNA synthesis, was needed for the first developmental stages. The activity of two enzymes involved in the synthesis of N-acetylglucosamine increased markedly during germination. THis increase was inhibited by both 5-fluorouracil and cycloheximide; this suggested that those enzymes were synthesized on mRNA formed during germination.

Acetylglucosamine↗
PubMed1981

Trehalase activity in extracts of Phycomyces blakesleeanus spores following the induction of germination by heat activation.

The heat activation of trehalase in extracts of sporangiospores of Phycomyces blakesleeanus, following the induction of germination by heat activation and the gelatinization of potato starch granules were studied under different conditions in order to discriminate between several phenomena as possible triggers in the activation of trehalase. Short-chain alcohols (from methanol to pentanol) lower the activation temperature of trehalase while long-chain alcohols (from heptanol to nonanol) raise it. Short-chain alcohols also lower the gelatinization temperature of potato starch granules, while long-chain alcohols, hexanol and heptanol have hardly any influence on the gelatinization temperature. Octanol raises the gelatinization temperature. More polar phenols lower the activation temperature of trehalase, while more apolar phenols will raise it. The gelatinization temperature of starch granules is more lowered by the polar polyphenols than by the more apolar phenols. The effect of high pressure on starch gelatinization was investigated in order to compare data from such a model system with the data on trehalase activation. The gelatinization temperature of starch granules is shifted upwards with about 3-5K/1000 atm (1.013 X 10(5) kPa). Pressures higher than 1500 atm do not further increase the gelatinization temperature. However, no reversal of the effect, as occurs with protein conformational changes, is seen with pressure up to 2500 atm. Also for trehalase activation we find a continuous upward shift of the activation temperature with about 5-9K/1000 atm. These data are in agreement with a thermal transition in a polysaccharide matrix, being the trigger in the heat activation of trehalase.

Ethanol↗
PubMed1977

The effect of gamma radiation on breaking of dormancy in Phycomyces spores.

Dormant sporangiospores of Phycomyces blakesleeanus were activated by Cs-137 gamma rays. After a dose of about 300 krad, between 80 and 90% of the spores germinated normally. However, further development of the mycelium was inhibited. Even with dry spores a partial activation was obtained. Activation by gamma rays was not accompanied by an increase in trehalase activity.

Cesium Radioisotopes↗
PubMed1989

Electrophoretic analysis of proteins from Phycomyces mutants with abnormal tropisms.

Certain phototropism mutants of Phycomyces blakesleeanus show defective bending responses (tropisms) to stimuli besides light, such as gravity, wind, and barriers. These so-called "stiff" mutants are affected in four genes (madD to madG). Using two-dimensional gel electrophoresis, we have analyzed polypeptides from microsomal and soluble fractions obtained from the wild type, four single mutants, and six double mutants affected in all pairwise combinations of the four genes. Consistent differences in spot patterns for madE and madF mutants were found in microsomal fractions but not in soluble fractions. In madE mutants, two spots designated E1 (52 kDa, pI 6.65) and E2 (50 kDa, pI 6.65) were altered. E1 appeared denser in the wild type than in the madE mutants, while the reverse was true for E2. The spots E1 and E2 are probably under regulatory control by madE, perhaps involving posttranslational modification. A protein spot, F1 (53 kDa, pI 6.1), was present on the wild-type gels but absent from all gels for madF mutants. The F1 polypeptide probably represents the madF gene product.

Electrophoresis, Gel, Two-Dimensional↗
PubMed1976

Recombination between mutants of Phycomyces with abnormal phototropism.

Sexual crosses were studied between mutants of Phycomyces blakesleeanus with abnormal phototropism (phenotype mad). Recombination frequencies were determined among five genes madA to madE. No clear evidence was found for linkage between any of the genes. Inconsistent results in crosses involving madC are attributed to nonisogenicity between the particular strains used. One mad strain was discovered to be a double mutant. A new gene, tentatively designated madG, was segregated from a cross involving that strain.

Crosses, Genetic↗
PubMed1988

System analysis of Phycomyces light-growth response with Gaussian white noise and sum-of-sinusoids test stimuli.

The sporangiophore (fruiting body) of the fungus Phycomyces modulates its elongation rate in response to changes in blue light intensity. This light-growth response of wild-type and behavioral mutant strains has been studied extensively by two methods of nonlinear system identification employing Gaussian white noise and sum-of-sinusoids test stimuli. Both methods are in the framework of the Wiener theory of nonlinear systems. The light-growth response is well described by the first-order Wiener G-functional; addition of the second-order functional improves the precision. The Wiener kernel of first-order resembles the light-growth response to a nonsaturating pulse stimulus. The second-order kernel indicates the nonlinear property of rectification. The kernels have been interpreted by system analysis methods in the frequency domain. A nonlinear dynamic model of the light-growth response has been developed from the kernels obtained by both methods. The model includes a nonlinear dynamic subsystem, including a squarer, followed by a linear dynamic subsystem (which, by itself, accounts for the first-order kernel). This parametric model has been used to evaluate light-growth response kernels under various conditions (viz. wavelength, temperature, and genetic strain). The kernels of single and double mutants have been analyzed jointly with a nonparametric model to reveal interactions among the products of eight genes that influence the light-growth response. The extent of interactions found suggests that these gene products function together in a molecular complex.

Light↗