Abnormal genesis of basidiospores in a mutant stock ofSchizophyllum commune FR

The Kniep stock subcultured for more than 45 years in the Centraal Bureau voor Schimmelcultures at Baarn, Netherlands, contains a burden of morphological and biochemical mutations. It is said to have been originally a monokaryon; something has obviously happened to it: either it has been mislabeled, or it has become contaminated during its long period of subculturing.Our results obtained from outcrossing, indicate that the Kniep stock is a dikaryon. Even mycelia derived from single spores behave as dikaryons and heterokaryons. An explanation of the abnormal behaviour of monosporous mycelia isolated from the Kniep stock is given in terms of impaired distribution of the nuclei from the basidia to the basidiospores, resulting in mono-, di-, and heterokaryotic basidiospores.     

Regions in theZ5 mating gene ofSchizophyllum commune involved in Y-Z binding and recognition

The Aα mating locus of the woodrotting fungusSchizophyllum commune encodes two multiallelic genes,Y andZ, which regulate the A-pathway of development. TheY alleles contain a homeobox, suggesting that the Y proteins may be DNA-binding regulatory proteins. During mating, development is induced when Y from one mating partner interacts with Z from the other mating partner; self combinations of Y and Z are inactive. Two-hybrid analyses indicate that nonself combinations of Y and Z form heteromultimers and self combinations do not. To understand Y-Z binding and self- nonself recognition further we used mutagenesis and chimeras to identify regions in one allele ofZ(Z5) that are involved in these processes. Here we report the results, which broadly define regions in Z5 that are essential for activity, Y-Z binding and Z5 allelic specificity.     

Erythritol metabolism in wild-type and mutant strains of Schizophyllum commune

Erythritol uptake and metabolism were compared in wild-type mycelium and a dome morphological mutant of the wood-rotting mushroom Schizophyllum commune. Wild-type mycelium utilized glucose, certain hexitols, and pentitols including ribitol, as well as d-erythrose, erythritol, and glycerol as sole carbon sources for growth. The dome mutant utilized all of these compounds except d-erythrose and erythritol. Erythritol- or glycerol-grown wild-type mycelium incorporated erythritol into various cellular constituents, whereas glucose-grown cells lagged considerably before initiation of erythritol uptake. This acquisition was inhibited by cycloheximide. Dome mycelium showed behavior similar to wild-type in uptake of erythritol after growth on glucose or glycerol, except that erythritol was not further catabolized. Enzymes of carbohydrate metabolism were compared in cell extracts of glucose-cultured wild-type mycelium and dome. Enzymes of hexose monophosphate catabolism, nicotinamide adenine dinucleotide (NAD)-dependent sugar alcohol dehydrogenases, and reduced nicotinamide adenine dinucleotide phosphate (NADPH)-coupled erythrose reductase were demonstrated in both. The occurrence of erythrose reductase was unaffected by the nature of the growth carbon source, showed optimal activity at pH 7, and generated NAD phosphate and erythritol as products of the reaction. Glycerol-, d-erythrose-, or erythritol-grown wild-type mycelium contained an NAD-dependent erythritol dehydrogenase absent in glucose cells. Erythritol dehydrogenase activity was optimal at pH 8.8 and produced erythrulose during NAD reduction. Glycerol-growth of dome mycelium induced the erythritol uptake system, but a functional erythritol dehydrogenase could not be demonstrated. Neither wild-type nor dome mycelium produced erythritol dehydrogenase during growth on ribitol. Erythritol metabolism in wild-type cells of S. commune, therefore, involves an NADPH-dependent reduction of d-erythrose to produce erythritol, followed by induction of an NAD-coupled erythritol dehydrogenase to form erythrulose. A deficiency in erythritol dehydrogenase rather than permeability barriers explains why dome cannot employ erythritol as sole carbon source for mycelial growth.     

Ultrastructural differences between wall apices of growing and non-growing hyphae ofSchizophyllum commune

Summary Newly synthesized chitin at the hyphal apex ofSchizophyllum commune was shown to be highly susceptible to chitinase degradation and solubilization by dilute mineral acid. With time this chitin became gradually more resistant to these treatments. With a combination of the shadow-cast technique and electron microscopic autoradiography it could be shown that this process occurred as the newly synthesized chitin moved into subapical parts of growing hyphae but also in non-growing apices which had ceased growth after incorporation of theN-acetyl[6-³H]glucosamine. These results are in agreement with a model which explains apical morphogenesis by assuming that the newly synthesized wall material at the apex is plastic due to the presence of individual polymer chains but becomes rigidified because of subsequent physical and chemical changes involving these polymers.Dedicated to Dr. A.Quispel, Professor of Botany at the University of Leiden, on occasion of his retirement.     

Repair of UV-induced damage in wild-type and mutant strains of Schizophyllum commune

The basidiomycete fungus Schizophyllum commune was found to have both photo-repair and dark-repair systems for UV-induced damage. Three UV-sensitive mutants were isolated and characterized for ability to repair UV-induced damage in light and dark, and for cross-sensitivity to caffeine and methyl methanesulfonate. Two of the mutants were damaged, to different extents, in their capacity for excision repair; one of these mutants was also probably damaged in post-replication repair. The third mutant was damaged only in post-replication repair.     

Entry of methotrexate into Streptococcus pneumoniae: a study on a wild-type strain and a methotrexate resistant mutant

Entry of methotrexate (MTX) into the folate prototrophic bacterium Streptococcus pneumoniae was poorly inhibited by folate or its natural derivative folinic acid, suggesting that if MTX is transported via a folate transporter, the affinity of that transporter for MTX is higher than for folate. In the range of concentrations tested, MTX uptake was non-concentrative and decreased in ATP-depleted bacteria. When the external concentration of MTX was increased from 1 X 10(-7) M to 1 X 10(-6) M, uptake became saturated and was insensitive to ionophores. However when external MTX concentrations were increased to 1 X 10(-5) M, uptake increased linearly, and was inhibited by the ionophores carbonyl cyanide m-chlorophenylhydrazone (CCCP) and valinomycin, suggesting that the process was energized by the protonmotive force (delta p) at this concentration. A model for MTX entry in S. pneumoniae is proposed with respect to these results. The high level of resistance to MTX of the nonsense mutant amiA9 cannot be entirely explained by a decrease in MTX uptake.     

Griseofulvin-induced alterations in site of dividing nuclei and structure of septa in a dikaryon ofSchizophyllum commune

Summary Ultrastructural study of a dikaryon of the basidiomyceteSchizophyllum commune showed that treatment with griseofulvin affected the site of the dividing nuclei and the location and structure of the septa. The microtubules were considered to be the primary target of griseofulvin, since they participate in nuclear division and movement in the hyphae, and their assembly is known to be in other organisms than fungi inhibited by griseofulvin. It is pointed out that dikaryotic hyphae with two nuclei and a clamp connection per cell are more sensitive indicators of the effect of griseofulvin than homokaryotic hyphae, whose structure is less complex.     

Ultrastructure and localization of wall polymers during regeneration and reversion of protoplasts ofSchizophyllum commune

Summary Cell-wall regeneration and reversion of protoplasts ofSchizophyllum commune were investigated using electron microscopic methods and X-ray diffraction.After 3 hours of regeneration protoplasts have formed a loosely organized wall which does not react with Thiéry's stain for periodic acid sensitive carbohydrates. This wall largely consists of chitin microfibrils which are adpressed to the plasmalemma and which are covered by loose aggregates of alkali-soluble S-glucan (-1,3-glucan). Both components are microcrystalline, at least partly. Walls formed in the presence of polyoxin D only consist of thick loose fibers of S-glucan.From 3 hours onward the inner chitin microfibrils of the wall of the primary cells become embedded in alkali-insoluble material that stains heavily with the Thiéry reagent and probably is similar to the R-glucan of the mature wall (i.e., -1,3--1,6-glucan). The outer chitin microfibrils remain free of this matrix and are covered by S-glucan only.Bud-like structures that arise have the same wall architecture as the primary cells,i.e., only the inner chitin microfibrils are embedded in R-glucan and the S-glucan forms a fluffy coat. The walls of hyphal tubes that arise are distinct, however, in that all chitin microfibrils are embedded in R-glucan and the S-glucan forms a compact coat.Cytoplasmic vesicles are sparse in primary cells except at the sites of emergence of budlike structures and hyphae. They continue to be present in the apex of growing hyphae.     

Abnormal zinc metabolism in unilateral maldescended testes of a mutant rat strain

The status of zinc in a mutant rat strain with heritable maldescended testes was examined. In rats with unilateral maldescended testis, the ectopic testis consistently had decreased zinc content (121.0 +/- 23.0 micrograms zinc/g dry wt), while the eutopic testis had zinc content similar to that of normal rats (182.0 +/- 5.0 micrograms zinc/g dry wt). Uptake of zinc by the ectopic testis was comparable to normal. Sephadex gel chromatography showed greatly reduced zinc content of one of the endogenous zinc binding fractions with a mol wt of 30,000 of cytosol of the ectopic testis in spite of a near normal protein content. Incorporation of zinc-65 into this fraction was also shown to be greatly reduced in ectopic testis. Sodium dodecylsulphate-polyacrylamide gel electrophoresis demonstrated that a protein of 23,000 Da was greatly reduced in quantity. This 23-kDa protein in ectopic testis may play a role in reduced testicular function of the ectopic testis.     

Light microscope and electron microscope alkaline phosphatase cytochemistry of rat bone marrow leukocytes.

An enzyme cytochemical method yielding an osmiophilic reaction product, visible at both the light and electron microscope levels, has been applied to the study of alkaline phosphatase in rat bone marrow cells. The enzyme is present in both eosinophils and, in much smaller amounts, in neutrophils. In both cases it is present on the plasma membrane, and in eosinophils intracellular aggregations of reaction product are also seen. The specific granules in both cell types fail to react and the enzyme is first detectable at the promyelocyte stage. Thus the enzyme is demonstrable before specific granule formation begins in the neutrophil, indicating that they are not a significant site of alkaline phosphatase activity in the rat.     

Occurence of microtubules and microfilaments,and origin of septa in dikaryotic hyphae ofSchizophyllum commune

Summary In an electron microscopic study on the dikaryotic hyphae ofSchizophyllum commune, microtubules were observed during the nuclear division, and close to the non-dividing nuclei of apical cells and older cells. Microtubules of the spindle were connected with semicircular bodies at nuclear poles. Microfilaments were detected in the distal part of the apical cells. Vesicles similar to those in the tips of the hyphae occured also at the sites of septa formation. The occurrence of microtubules and the structure of semicircular bodies are compared with those in other basidiomycetes. It is suggested that vesicles are involved in the primary growth of the septal cross wall.     

Molecular cloning of RNAs differentially expressed in monokaryons and dikaryons ofSchizophyllum commune in relation to fruiting

A complementary DNA library cloned in pBR327 was prepared from RNA of a fruiting dikaryon of the basidiomyceteSchizophyllum commune. An improved colony-lysis method led to the isolation of complementary DNA clones hybridizing to nine different RNAs abundantly present in the fruiting dikaryon but present in very low or undetectable levels in the monokaryons. One of these RNAs, measuring 580 nucleotides, was exceptional in contributing more than 3% to the mRNA mass of the fruiting dikaryon. A role of the detected dikaryon-specific RNAs in fruiting was suggested by (i) their absence or low concentration in vegetatively growing monokaryons or the dikaryon, (ii) their steep increase in concentration during fruit-body formation in the dikaryon, (iii) a higher concentration of most of these RNAs in the fruit-body initials than in the supporting mycelium, and (iv) their prevalence in full-grown fruit bodies. The “fruiting-specific” RNAs were also present in a suspension-grown dikaryon, in the absence of fruiting, but in concentrations less than 10% of those in surface-grown fruiting dikaryon. The level of expression of these RNAs in the monokaryons was even lower or undetectable. Since the dikaryon and the monokaryons are otherwise coisogenic, it appears that the presence of two different incompatibility genes in the dikaryon allows for a low level of expression of the “fruiting genes” even in the absence of fruiting, whereas the genes are fully expressed under conditions permitting fruit-body formation.     

Bacteriophage that can distinguish between wild-type Rhizobium japonicum and a non-nodulating mutant

A bacteriophage (phage TN1) that lyses Rhizobium japonicum 3I1b110 was isolated from Tennessee soil. Structurally, this phage resembles the Escherichia coli phage T4, having an icosahedral head (47 by 60 nm) and a contractile tail (17 by 80 nm). An interesting feature of this phage is that it lyses all of the symbiotic defective mutants derived from R. japonicum 3I1b110 that were tested, except one, mutant strain HS123. Mutant strain HS123 is a non-nodulating mutant that is defective in attachment to soybean roots. Since Rhizobium attachment to host roots is thought to be mediated by a specific cell surface interaction, it is likely that mutant strain HS123 is defective in some way in its cell surface. Mutant strain HS123 bound soybean lectin to the same extent as the wild type as measured by the binding of tritium-labeled lectin. Phage TN1 did not attach to the surface of strain HS123, nor did cells of strain HS123 inactivate phage TN1. A hot phenol-water cell extract from the wild-type inactivated phage TN1, whereas a similar cell extract from mutant HS123 did not. Capsular polysaccharide isolated from mutant or wild type did not inactivate the phage. Capsular polysaccharide and exopolysaccharide from the mutant and wild type do not differ in sugar composition. These results indicate that capsular polysaccharide may not play a role in attachment to the plant root surface and that other cell wall components may be important.     

Energetics of wild-type and mutant multidrug resistance secondary transporter LmrP of Lactococcus lactis

LmrP, a proton/multidrug antiporter of Lactococcus lactis, transports a variety of cationic substrates. Previously, two membrane-embedded acidic residues, Asp142 and Glu327, have been reported to be important for multidrug transport activity of LmrP. Here we show that neither Glu327 nor Asp142 is essential for ethidium binding but that Glu327 is a critical residue for the high affinity binding of Hoechst 33342. Substitution of these two residues, however, negatively influences the transport activity. The energetics of transport was studied of two closely related cationic substrates ethidium and propidium that carry one and two positive charges, respectively. Extrusion of monovalent ethidium is dependent on both the electrical membrane potential (Deltapsi) and transmembrane proton gradient (DeltapH), while extrusion of propidium predominantly depends on the DeltapH only. The LmrP mutants D142C and E327C, however, mediate electroneutral ethidium extrusion, but are unable to mediate DeltapH-dependent extrusion of propidium. These data indicate that Asp142 and Glu327 are involved in proton translocation.     

Light and electron microscope study of a new species of Thelohania (Microsporida) in the shrimp Pandalus jordani

Thelohania butleri n. sp. was found in cells of skeletal muscles of the shrimp Pandalus jordani, from Queen Charlotte Sound, British Columbia, Canada. Sporulation stages were studied with the light and the electron microscope. Earliest stages were small and apparently uninucleate. Next were small diplokaryotic cells that possibly arose by fusion of the former. These enlarged and underwent sporogony. Sporogony was a series of three binary divisions, each producing unikaryotic cells. There was no sporogonial plasmodium. The spore was ovoid, 4.8 × 3.1 μm (stained), with a large crescentic nucleus and rounded posterior vacuole. The polar filament was isofilar, doubly coiled, with about 10 turns. This species closely resembles the type T. giardi Henneguy. It is concluded that sporogony by means of three binary divisions and lack of a sporogonial plasmodium may be essential characters of the genus Thelohania Henneguy, 1982.     

Unusual structure of nuclei in a B–mutant strain of Schizophyllum commune with intercellular nuclear migration

Serial sections of multinucleate hyphae of a B–mutant strain of Schizophyllum commune , in which intercellular nuclear migration takes place, revealed appendages attached to nuclei. The appendages were strand–like organelles surrounded by a membrane with an electron–dense content. In addition, the outer membranes of the envelopes of adjacent nuclei were often joined and surface sections of these nuclei showed fibrous material with only a few nuclear pores. These ultrastructural features have not been reported before in the vegetative hyphae of S. commune , and they are suggested to be necessary for intercellular nuclear movement. The appendages may create the force or may result from the force necessary for the movement of the nucleus, while the joining of the outer membranes of the nuclear envelopes makes possible simultaneous movement of several nuclei, although only one has an appendage. The fibrous material in the nuclear envelope may facilitate sliding of the nuclei through the cytoplasm.