Relationship of photocarotenogenesis to other behavioural and regulatory responses inPhycomyces

The effect of light on carotene accumulation was studied by analyzing the -carotene content of 4--old mycelia continuously exposed to illumination of different intensities. The wild-type, mutants defective in phototropism, mutants defective in carotene regulation, and newpic mutants specifically defective for photocarotenogenesis were examined. The results indicate that photocarotenogenesis depends on a single sensory pathway which shares its earlier steps (governed by genesmadA andmadB) with the sensory pathway for phototropism. It shares its later steps (probably governed by genescarA andpicB) with one of the pathways for carotene regulation, and includes at least one specific step (governed by genepicA) not known to be involved in other responses.     

A genetic map of Phycomyces blakesleeanus

Summary 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.     

Excess carotenoids disturb prospective cell-to-cell recognition system in mating responses ofPhycomyces blakesleeanus

Carotenogenic mutants ofPhycomyces, which accumulate excess β-carotene or its intermediates, always failed in zygospore development. No improvement occurred when such mutants were mated together with a helper wild type of the same mating type against the wild type of the opposite mating type. Addition of excess synthesized pheromone, trisporin B, also failed to improve the zygospore development, though the mating response was significantly activated in the early stages and abundant zygophores were formed. Exceptional acceleration of the zygospore development under these experimental conditions occurred in a regulatory albino mutant (carA), which does not accumulate excess intermediate carotenoids. Chemically- or genetically-induced ovarproduction of β-carotene or lycopene also inhibited the zygospore development. These results imply that the zygospore development ofPhycomyces is maximal when the intracellular amount of β-carotene is optimal (=wild type), and that pheromones act mainly in the early stages of mating, while other factors such as the cell-to-cell recognition system may also be involved in the later stages. Intracellular accumulation of excess β-carotene or its intermediates probably disturb such later-stage factors.     

Relationships among the biosyntheses of ubiquinone, carotene, sterols, and triacylglycerols in Zygomycetes

The Zygomycetes Phycomyces blakesleeanus and Blakeslea trispora are actual or potential sources of β-carotene, ergosterol, ubiquinone, edible oil, and other compounds. By feeding [¹⁴C]acetyl-CoA, L-[¹⁴C]leucine, or R-[¹⁴C]mevalonate in the presence of excess unlabeled glucose, we found that ubiquinone (the terpenoid moiety), β-carotene, and triacylglycerols were made from separate pools of all their common intermediates; the pools for ubiquinone and ergosterol were indistinguishable. Fatty acids were not labeled from mevalonate, showing the absence in these fungi of a shunt pathway that would recycle carbon from mevalonate and its products back to central metabolism. The overproduction of carotene in a Phycomyces mutant and in sexually mated cultures of Blakeslea modified the relative use of labeled and unlabeled carbon sources in the production of carotene, but not of the other compounds. We concluded that carotene, ubiquinone, and triacylglycerols are synthesized in separate subcellular compartments, while sterols and ubiquinone are synthesized in the same compartments or in compartments that exchange precursors. Carotene biosynthesis was regulated specifically and not by flow diversion in a branched pathway.     

DNA of Akodon (Rodentia,Cricetidae). I. Biophysical characterization

Buoyant density in CsCl, melting temperature, and G + C base content of the DNA from four species of Akodon (Rodentia, Cricetidae) were determined. The buoyant density values of 1.699–1.701 g/cm³ were in accordance with the data reported for other cricetids. No satellite bands were seen in neutral CsCl. The T _m values determined in 1 × SSC ranged from 86.2 to 87.0 C, which corresponds to G + C contents of 41.2–43.2%. There was good agreement in DNA base composition of the four species, although values were slightly higher in A. obscurus, suggesting a certain degree of interspecies variability.This study was supported by grants from Comisión de Investigaciones Científicas de la Provincia de Buenos Aires, Consejo Nacional de Investigaciones Científicas y Técnicas, and Organización de Estados Americanos.     

Isolation of unselected mutants of alkaline phosphatase in Escherichia coli through nitrosoguanidine comutation and comparison with natural variants

We have devised a general procedure to isolate enzymatic variants without selecting or screening for related phenotypic peculiarities of the organism. A high mutation rate at phoA, the structural gene for alkaline phosphatase, is found among N-methyl-N'-nitro-N-nitrosoguanidine-induced proC revertants of Escherichia coli. About 1.6% of such revertants lack alkaline phosphatase, and many others exhibit altered enzyme parameters. Three mutants studied in detail had full enzyme activity but differed from the wild type in electrophoretic mobility, thermostability, and, in one case, optimum pH for enzyme activity. Four other phosphatase variants were discovered in a survey of 50 natural E. coli isolates; their electrophoretic mobility and thermostability were different from those of the wild type. Natural and induced enzyme variants are similar enough to suggest the absence of strong selective pressures in natural populations.This work was supported by grants from the Fundación J. March and the Comisión Asesora para la Investigación Científica y Técnica.     

Kinetics of photoaccumulation of β-carotene in Phycomyces blakesleeanus

Upon carbon starvation the -carotene content of Phycomyces mycelium grown on minimal agar medium disappears with a time lag of about 90 min and a T_1/2 of 68–75 min. If continuous light is given 2 h after starvation, there is an increase in -carotene content with respect to the dark control. This increase has a time lag of 20–25 min. The fluence rate-response curve of wt is biphasic and two mutants in the gene madA (madA7, madA35) and in the gene madB (madB101, madB104) have higher thresholds than wt; madB mutants are blinder than madA mutants. Only blue light is effective and we suggest that it has an effect solely on the catabolism of -carotene.Abbreviations D dark - L light - wt wild type     

Accumulation of carotenoids in structural and regulatory mutants of the bacterium Myxococcus xanthus

Summary Accumulation of carotenoids in Myxococcus xanthus is absolutely dependent on illumination with blue light. We report the analysis of the carotenoids of dark- and light-grown cultures of the wild type and several previously characterized mutants. A carR mutant produces the same carotenoids in the dark as the wild type grown in the light. This agrees with previous evidence indicating that the carR gene codes for a general negative regulator of the system. A cis-dominant mutation in the gene carA causes constitutive expression of the light-inducible gene carB, which is linked to carA. In the dark, the carA mutant produces high levels of phytoene, the first C₄₀ colourless carotenoid precursor; in the light, it produces the same carotenoids as the wild type. Since a mutation in carB blocks accumulation of phytoene, we propose that carB, and probably other linked genes also controlled by carA, code for enzymes involved in the synthesis of phytoene. This is virtually the only carotene accumulated by strains mutated in the gene carC, which is unlinked to the others. Thus carC codes for phytoene dehydrogenase, the enzyme that converts phytoene into coloured carotenoids. The results presented here also provide evidence for control of carotenogenesis by an endproduct that is independent of the blue light effect.     

Phycomyces: Phototropism and light-growth response to pulse stimuli

The relationship between phototropism and the light-growth response of Phycomyces blakesleeanus (Burgeff) sporangiophores was investigated. After dark adaptation, stage-IVb sporangiophores were exposed to short pulses of unilateral light at 450 nm wavelength. The sporangiophores show a complex reaction to pulses of 30 s duration: maximal positive bending at 3·10^-4 and 10^-1 J m^-2, but negative bending at 30 J m^-2. The fluence dependence for the light-growth response also is complex, but in a different way than for phototropism; the first maximal response occurs at 1.8·10^-3 J m^-2 with a lesser maximum at 30 J m^-2. A hypertropic mutant, L85 (madH), lacks the negative phototropism at 30 J m^-2 but gives results otherwise similar to the wild type. The reciprocity rule was tested for several combinations of fluence rates and pulse durations that ranged from 1 ms to 30 s. Near the threshold fluence (3·10^-5 J m^-2), both responses increase for pulse durations below 67 ms and both have an optimum at 2 ms. At a fluence of 2.4·10^-3 J m^-2, both responses decrease for pulse durations below 67 ms. The hypertropic mutant (madH), investigated for low fluence only, gave similar results. In both strains, the time courses for phototropism and light-growth response, after single short pulses of various durations, show no clear correlation. These results imply that phototropism cannot be caused by linear superposition of localized light-growth responses; rather, they point to redistribution of growth substances as the cause of phototropism.     

(−) Mating Type-Specific Mutants ofPhycomycesDefective in Sex Pheromone Biosynthesis

Sutter, R. P., Grandin, A. B., Dye, B. D., and Moore, W. R. 1996. (−) Mating type-specific mutants ofPhycomycesdefective in sex pheromone biosynthesis.Fungal Genetics and Biology20,268–279. We have isolated the first mating type-specific mutants in mucoraceous fungi. Both mutants inPhycomyces blakesleeanusappear to be defective in the same gene. The gene, present in both mating types, is necessary only in cultures of the (−) mating type. The gene codes for an enzyme in sex pheromone biosynthesis. The pheromone precursor made by the mutants is detectable only in cross-feeding experiments. The biological and solubility properties of the precursor suggest the precursor is 4-dihydrotrisporin, a metabolite of β-carotene. Separate studies with β-carotene-deficient mutants and Compound-P, a new chemically synthesized precursor of the pheromones, imply the constitutive level of enzymes for pheromone biosynthesis inPhycomycesis extremely low. In comparison, the level of enzymes for pheromone conversion to trisporic acid is higher. The mating type-specific mutants also catalyze the conversion of (+) pheromone to trisporic acid. This finding was unexpected because literature models predicted this reaction was catalyzed by the same enzyme which catalyzed the conversion of 4-dihydrotrisporin to (−) pheromone—a reaction missing in the (−) mating type-specific mutants. Thus, we propose a revised model for trisporic acid biosynthesis.     

High-and low-intensity photosystems in Phycomyces phototropism: Effects of mutations in genes madA,madB, and madC

Sporangiophores of Phycomyces blakesleeanus Burgeff that have been grown in darkness and are then suddenly exposed to unilateral light show a two-step bending response rather than a smooth, monotonic response found in light-adapted specimens (Galland and Lipson, 1987, Proc. Natl. Acad. Sci. USA 84, 104–108). The stepwise bending is controlled by two photosystems optimized for the low-and high-intensity ranges. These two photosystems have now been studied in phototropism mutants with defects in genes madA, madB, and madC. All three mutations raise the threshold of the low-intensity (low-fluence) photosystem by about 10⁶-fold and that of the high-intensity (high-fluence) system by about 10³-fold. Estimates for the light-adaptation time constants of the low-and high-intensity photosystems show that the mutants are affected in adaptation. In the mutants, the light-adaptation kinetics are only slightly affected in the low-intensity photosystem but, for the high-intensity photosystem, the kinetics are considerably slower than in the wild type.Abbreviations WT wild type     

Electrophoretic analysis of proteins fromPhycomyces mutants with abnormal tropisms

Certain phototropism mutants ofPhycomyces 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 tomadG). 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 formadE andmadF mutants were found in microsomal fractions but not in soluble fractions. InmadE 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 themadE mutants, while the reverse was true for E2. The spots E1 and E2 are probably under regulatory control bymadE, 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 formadF mutants. The F1 polypeptide probably represents themadF gene product.This work was supported by research grants to E.D.L. from the National Science Foundation (DMB-8316458 and DMB-8704602) and an equipment grant to C.H.T. from the Syracuse University Senate Research Committee.     

Form II of D1 is important during transition from standard to high light intensity in Synechococcus sp. strain PCC 7942

In Synechococcus sp. strain PCC 7942 the D1 protein of Photosystem II is encoded by a multigene family; psbAI encodes Form I of D1 whereas both psbAII and psbAIII encode Form II. The psbA genes are differentially regulated in response to changes in light intensity, such that psbAI expression and Form I predominate at standard light intensity, whereas psbAII and psbAIII are induced at high light intensity, causing insertion of Form II into the thylakoids. The present study addressed whether high-light induced Form II is important for Synechococcus cells during adaptation to high light intensity. Wild-type Synechococcus, and mutants which produce only Form I (R2S2C3) or only Form II (R2K1), were co-cultured at standard light (130 E · m^–2 · s^–1) and then shifted to high light (750 E·m^–2·s^–1). Measurement of the proportion of each cell type at various time intervals revealed that the growth of R2S2C3, which has psbAII and psbAIII inactive, and thus lacks Form II, is transiently impaired upon shift to high light. Both mutants R2S2C3 and R2K1 maintained normal levels of psbA messages and D1 protein under standard and high light through an unknown mechanism that compensates for the inactive psbA genes. Thus, the impairment of R2S2C3 at high light is not due to a deficiency of D1 protein, but results from lack of Form II. We discounted the influence of possible secondary mutations by re-creating the psbA-inactivated mutants and testing the newly isolated strains. We conclude that Form II of D1 is intrinsically important for Synechococcus cells during a critical transition period after exposure to high light intensities.     

Electrophoretic analysis of proteins from night-blind mutants of Phycomyces

Using two-dimensional gel electrophoresis, we have analyzed proteins from a plasma membrane-enriched fraction from Phycomyces sporangiophores. Specifically, we have compared gels for night-blind mutants and a wild-type strain to find proteins involved in the early steps of the sensory transduction chain for phototropism. In the gels for a mutant affected in the gene madA, a protein spot [51 kilodaltons (kdal) and pI 6.35] appears that is absent from the wild-type and the other mad mutants. Mutants affected in either of two madB alleles lack a protein spot (57 kdal and pI 6.6) that is present in the wild-type and all other mad strains; this spot probably represents the madB gene product. In some madC mutants, two spots (59 kdal, pI 6.5, with a covalently linked flavin; and 50 kdal, pI 6.4) are absent; however, in other madC strains, one or both of these spots are present. These four protein spots that are altered in madA, madB, and madC mutants may represent components of the photoreceptor complex responsible for phototropism in Phycomyces.This work was supported in part by an equipment grant to JAP from the Syracuse University Senate Research Committee, research grants to EDL from the National Science Foundation (PCM-8003915 and DMB-8316458), and a fellowship to EDL from the Alfred P. Sloan Foundation.     

End-product regulation of carotenogenesis in Phycomyces

Wild-type Phycomyces blakesleeanus synthesizes the yellow pigment, beta-carotene. Colour mutants exhibit various alterations in the biosynthesis of beta-carotene or in its regulation. The presence of certain chemicals in the medium stimulates carotenogenesis in the wild type. We attribute different mechanisms of action to agents which stimulate or fail to stimulate different sets of mutants; this is the case of retinol and dimethyl phthalate. Dimethyl phthalate and veratrol are active on the same mutants, and therefore are likely to act in the same way. The main regulation of carotenogenesis, end-product inhibition, does not operate in the mutants of certain genes; these mutants are indifferent to retinol. By using a collection of retinoids we conclude that their action depends on their structural similarity to a part of the beta-carotene molecule. From these and other observations we propose that end-product inhibition of the pathway is mediated by a complex of beta-carotene and two gene products and that the retinoids compete with beta-carotene and prevent end-product inhibition.Deceased     

Transformation of Phycomyces with a bacterial gene for kanamycin resistance

Summary Phycomyces protoplasts transformed with a plasmid containing the bacterial gene for kanamycin resistance grow in the presence of G418, a kanamycin analogue. The plasmid also contains a Phycomyces DNA sequence that supports autonomous replication in yeast. We obtained about 250 transformants per microgram DNA or one per 5000 viable protoplasts. The transformant phenotype is retained under selective conditions and lost in the majority of the vegetative spores. Recovered plasmids and Southern analysis indicate that the plasmid probably replicates autonomously in Phycomyces.     

Complementation between mutants of Phycomyces deficient with respect to carotenogenesis

Summary White and red mutants of Phycomyces, derived from two independent wild types (yellow) by mutagenesis using nitrosoguanidine, either in a single step (26 white, 5 red mutants), or in two steps (10 white mutants, from one of the red mutants) were studied with respect to complementation in heterokaryons. The tests clearly establish the involvement of three and only three genes, here named carA, carB, and carB. The carA and the carR mutants are white, the carA mutants do not accumulate phytoene, the carB mutants do. The carR mutants are red and accumulate lycopene. The two step mutants are either carA and carR, or carB and carR double mutants. A few of the white mutants obtained in a single mutagenization step are affected in carA and carR. They may be polar mutants in an operon or accidental double mutants.     

Colloid droplets in the magnocellular secretory neurons of the reptilian hypothalamus: An immunocytochemical and lectin-histochemical study

Summary The immunocytochemical and lectin-binding properties of the magnocellular neurosecretory neurons in the hypothalamus of 2 reptilian species, the snake Natrix maura and the lizard Liolaemus cyanogaster, were investigated. Particular attention was paid to the secretory droplets present in these neurons. Antisera against bovine neurophysins I+II, arginine-vasotocin, and mesotocin were used. The following lectins were applied: concanavalin A (Con A), wheat-germ agglutinin (WGA), and Limax flavus agglutinin (LFA). Adjacent 1-m-thick methacrylate sections were used to investigate the same secretory neuron and the same colloid droplets with all three antisera and all three lectins. Several sections were treated with trypsin and urea before immunostaining or lectin binding. Con A bound to both vasotocin- and mesotocin-immunoreactive neurons, WGA exclusively to vasotocin neurons; neither of these neurons reacted with LFA. The colloid droplets were present in vasotocin neurons but absent in the mesotocin neurons. These secretory droplets showed an affinity for Con A but not for WGA, and reacted with antisera against neurophysins and vasotocin. In Natrix maura, the colloid droplets became reactive with Con A and the antisera used only after pretreatment of the sections with trypsin and urea. Within the hypothalamo-neurohypophyseal system, antiserum against vasotocin and WGA revealed the same fiber bundles. It is concluded (i) that in reptiles the vasotocin-neurophysin precursor is glycosylated, (ii) that vasotocin neurons have the exclusive capacity to form colloid droplets, and (iii) that these droplets are an intracisternal (RER) storage form of the vasotocin-neurophysin precursor.This work was partially supported by Grants BOJA 27/9/88 from the Dirección General de Universidades e Investigación de Junta de Andalucía and DGICYT PB87-0710 from the Comisión Interministerial de Ciencia y Tecnología, Madrid, to P.F.-LL.; and Grant 89-01 from the Dirección de Investigaciones, Universidad Austral de Chile, to E.M.R.     

Ionenabhängige Keimungsaktivierung der Sporangiosporen vonPhycomyces blakesleeanus undP. nitens,demonstriert an verschiedenen Stämmen

Zusammenfassung Es wurde der Einfluß der Kationen Na⁺, K⁺, Ca²⁺ und NH ₄ ⁺ in Verbindung mit dem Anion Acetat auf die Keimungsrate einigerPhycomyces-Stämme (P. blakesleeanus 1+, 2-, 11+ und 18- Sammlung Halbsguth; 188+ und 189-Sammlung Baarn;P. nitens 304+ und 305-Sammlung Baarn) hin untersucht. Es konnte gezeigt werden, daß den Kationen eine wesentliche Rolle im Aktivierungsprozeß zukommt. Förderlich wirkten sich vor allem K⁺ und Ca²⁺ Ionen aus. NH ₄ ⁺ wies keine sichtbar fördernde Wirkung auf, und dem Na^- Ion kann eine hemmende Wirkung im besonderen für dieP. blakesleeanus-Stämme 2-, 188+, 189- zugeschrieben werden. Eine vergleichbar empfindliche Reaktion zeigteP. nitens gegenüber Ca²⁺-Ionen, jedoch nur, wenn höhere Konzentrationen verwendet wurden. Allgemein waren dieP. nitens-Stämme 304+, 305- in ihrer Aktivierbarkeit nicht in dem Maße von den einzelnen Kationen abhängig wie dieP. blakesleeanus-Stämme.Ferner wurde die Wirkung der Wärmeaktivierung auf die Sporenkeimung geprüft. Die günstigste Dauer der Wärmeaktivierung bei 50°C lag für dieP. blakesleeanus-Stämme bei 5 min; dieP. nitens-Stämme vertrugen noch eine Zeit von 40 min (50°C) ohne erkennbare Schädigung.Die Aktivierbarkeit der Sporangiosporen in Abhängigkeit vom Aktivierungsagens (Acetat bzw. Wärme) zeigte deutliche Unterschiede zwischen denP. blakesleeanus- undP. nitens-Stämmen.

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Activating of germination dependent on lons of sporangiospores ofPhycomyces blakesleeanus andP. nitens, demonstrated on various strains

The influence of cations Na⁺, K⁺, Ca²⁺ and NH ₄ ⁺ in connection with anion acetate on the rate of germination was studied for severalPhycomyces strains (P. blakesleeanus 1+, 2-, 11+, 18- collection Halbsguth; 188+, 189- collection Baarn;P. nitens 304+ and 305- collection Baarn). Cations played an important role during the activating process; NH ₄ ⁺ did not further the effect. The Na⁺ ion inhibited. The inhibition was marked onP. blakesleeanus strains 2-, 188+ and 189-. A comparable sensitive reaction ofP. nitens was apparent towards Ca²⁺ ions, when higher concentrations were used. Generally activations ofP. nitens strains 304+ or 305- were not influenced by the different cations as much as were theP. blakesleeanus strains.Furthermore the effect of heat activation on spore germination was tested. The most favourable time period for heat activation at 50°C was 5 min for theP. blakesleeanus strains, while theP. nitens strains tolerated a period of 40 min at 50°C without any detectable damage (90% germinated spores).The ability ofPhycomyces sporangiospores to be activated showed differences between theP. blakesleeanus andP. nitens strains depending on the activating agent (heat or acetate).

     

Pollen morphology ofSonchus and related genera,and a general discussion

The pollen morphology of the taxa belonging to the generaAetheorhiza Cass.,Launaea Cass.,Reichardia Roth andSonchus L. in the Iberian Peninsula has been studied with light and electron microscopy. The pollen is 3(-4)-zonocolporate and echinolophate (without polar lacunae, but in general with prelacunae), with equatorial ridges and 15–20 lacunae: 3–4 poral, 6–8 abporal and 6–8 paraporal. Small to medium size, P × E = 19–36 × 23–42 µm; sometimes two different sizes have been found. Exine 3–9 µm thick and ornamentation microreticulate and echinate. The results clearly show the relationships between genera. Moreno-Socías, E., Mejías, J. A., Díez, M. J., 1994: Morfología polínica deLactuceae (Asteraceae) en la Península Ibérica, I.Lactuca y géneros relacionados. — Acta Bot. Malacitana.19: 103–113.