EVIDENCES OF COMMON-AB HETEROKARYOSIS IN SCHIZOPHYLLUM COMMUNE

Since preliminary experimental evidence for the existence of common-AB heterokaryons in Schizophyllum commune was inconclusive, experiments were performed to test for and to characterize these heterokaryons. Prototrophic mycelia regularly result from the growing together of pairs of mutant strains of identical mating type which carry non-allelic nutritional deficiencies. Since crossfeeding through a dialyzing Cellophane membrane does not occur, the prototrophic common-AB mycelia apparently have both parental nuclear types within a common cytoplasm. Hyphal tips isolated from the peripheries of these prototrophic mycelia usually are not prototrophic. The distributions of the parental nuclear types in the subcultured prototrophic mycelia, as revealed by grid sampling, are irregular in pattern and extent; these patterns probably reflect the nuclear ratios in the transfer inocula, as well as the distributions of nuclear types in various parts of the inocula. Since a common-AB prototroph typically consists of regions containing a single nuclear type as well as regions containing both nuclear types, marked fluctuations of the estimated nuclear ratio occur upon subculture, and many small transfer-inocula are not prototrophic as they contain only a single auxotrophic nuclear type. The patterns of nuclear distributions in prototrophic common-AB mycelia are probably maintained by the restriction of nuclear migration.     

Cloning and characterization of a gene (arpA) from Aspergillus oryzae encoding an actin-related protein required for normal nuclear distribution and morphology of conidiophores

We have isolated the arpA gene from Aspergillus oryzae as a homologue of the Neurospora crassa ro-4 gene. In N. crassa, mutations in the ro-4 gene, which encodes a major component of the dynactin complex Arp1, causes curling of hyphae and abnormalities in nuclear distribution. The arpA gene contains two introns and encodes a polypeptide of 381 amino acids, with a 78% sequence identity to the N. crassa Arp1. Overexpression of the arpA gene causes a defect in nuclear migration into elongating hyphae of germlings in A. oryzae. We constructed arpA disruptant strains of A. oryzae. The arpA null mutants showed poor growth and hyper-branched mycelia, as well as a nuclear distribution defect. Scanning electron microscopy revealed that the arpA null mutant has an aberrant conidiophore morphology with irregular phialides. Received: 26 January 1999 / Accepted: 22 July 1999     

The ultrastructure of septal pores and associated structures in the ascogenous hyphae and asci ofSordaria humana

Summary Septal pores and associated structures have been studied in ascogenous hyphae, croziers and asci ofSordaria humana by means of electron microscopy of serial and random sections. Pores exhibit variable structures from relatively simple pore caps to complex swollen rims with associated membrane cisternae. The simple types are found at the base of the ascogenous hyphae while the complex forms occur at the apex, in the croziers and in very young, presporulation asci. Post-sporulation asci contain a relatively simple type of pore structure. Cells which subtend the ascogenous hyphae exhibit both open and capped pores in their cross walls. Pore structures may be asymmetric in which case they show greater complexity on the side of the cross wall nearest to the apex or crozier. Membranous components of the complex pores are continuous with the endomembrane systems of the two adjacent cells and thus with the outer membranes of the nuclear envelopes. Membrane continuities may connect prefusion nuclei or fusion nuclei in penultimate cells, with nuclei of the stalk and terminal cells of croziers. Some speculation is presented as to the implication and possible roles of these structures in relation to cell differentiation within the ascogenous hyphae and croziers.     

Septal involvement in nuclear migration inSchizophyllum commune

Summary A system, utilizing thedwarf andpuff morphological mutants ofSchizophyllum commune, was devised to select specific hyphal segments that were in different stages of septal dissolution and nuclear migration. These stages were observed with the electron microscope. Direct evidence of the dissolution of complex septal during nuclear migration was obtained. Initially there was a disruption of the septal swelling, followed by erosion of the remaining cross wall. Complex septa were therepy converted to simple septa through which nuclei migrated. These septa were covered with secondary cell wall material. Following nuclear migration only vacuolate hyphae with remnant membrane structures remained. Occasionally, intact hyphae were observed within these vacuolate hyphae.     

Nuclear reassortment between vegetative mycelia in natural populations of the basidiomycete Heterobasidion annosum

Somatic incompatibility is not an absolute block to nuclear exchange between incompatible mycelia of the basidiomycete Heterobasidion annosum in vitro. Under laboratory conditions new heterokaryotic genotypes can be isolated from the gap between incompatible heterokaryons, and nuclear migration between pairs of heterokaryons grown in Petri dishes has been observed. In this study, we test the hypothesis of nuclear transfer and reassortment between heterokaryotic mycelia in natural populations of H. annosum. We developed six microsatellite markers to genotype nuclei populating 21 somatically incompatible mycelia of H. annosum isolated from a single stump of Picea abies, and found that the detected heterokaryons share nuclei; 10 of the nuclear haplotypes were found in more than one mycelium. In one isolate, four nuclear types were found in the same mycelium. These findings indicate that new heterokaryons can be formed as a result of nuclear reassortment between incompatible heterokaryotic mycelia in nature.     

Ascoma genotyping and mating type analyses of mycorrhizas and soil mycelia of Tuber borchii in a truffle orchard established by mycelial inoculated plants

Tuber borchii (the Bianchetto truffle) is a heterothallic Ascomycete living in symbiotic association with trees and shrubs. Maternal and paternal genotype dynamics have already been studied for the black truffles Tuber melanosporum and Tuber aestivum but not yet for T. borchii. In this study, we analysed maternal and paternal genotypes in the first truffle orchard realized with plants inoculated with five different T. borchii mycelia. Our aims were to test the persistence of the inoculated mycelia, if maternal and/or paternal genotypes correspond to inoculated mycelia and to assess the hermaphroditism of T. borchii. The mating type of each isolate as well as those of mycorrhizas, ascomata and extraradical soil mycelia was determined. Moreover, simple sequence repeat (SSR) profiles of maternal and paternal genotypes were assessed in 18 fruiting bodies to investigate the sexual behaviour of this truffle. The maternal genotypes of the fruiting bodies corresponded to those of the inoculated mycelia with only two exceptions. This confirmed that the inoculated mycelia persisted 9 years after plantation. As regards paternal partner, only two had the same genotype as those of the inoculated mycelia, suggesting hermaphroditism. Most of the new paternal genotypes originated from a recombination of those of inoculated mycelia.     

The nuclear pores of early meiotic prophase nuclei of plants

Summary Pollen mother cells at early meiotic prophase fromFritillaria lanceolata, F. mutica, Tulbaghia violacea, the lily “Formobel”,Triticum aegilopoides, T. dicoccoides, T. aestivum and synaptic and asynaptic forms ofT. durum were studied in thin sections with the electron microscope (a) in relation to distribution of nuclear pores (b) in respect of fine structure of the pore complex in those of the first four. The pores were distributed in random clusters during leptotene to pachytene in all plants, except in the two forms ofT. durum where there were either no pores or so few that they were not detectable. Probably correlated with this, the two membranes of the nuclear envelope were often widely separated and frequently sacculated. No pores were seen at leptotene in the part of the envelope to which, in theFritillarias and lily, the nucleolus was adpressed at this time. Evidence supporting a recent model which proposes that annuli are composed of three rings of eight granular subunits was obtained. These subunits as well as a dense central element, observed in most pores, were composed of filaments about 3 nm in diameter and evidently protein in character. There was evidence of a continuity between filaments in the central element and those in the rings of subunits which encircle the pore aperture at both the nuclear and cytoplasmic sides of the pore. In profiles of pores knobbed filaments were sometimes seen extending laterally from the pore wall into the perinuclear space at two sides. Questions concerning the role of the annulus are discussed. The author wish to thank Mr. R. F. Scott for construction to the model.     

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.     

Fractionation and Anti-tumor Activity of the Mycelia of Liquid-cultured Phellinus linteus

All the fractions of Phellinus linteus mycelia showed anti-tumor activity toward solid tumors planted in mice. The highest anti-tumor activity of 81.2% was observed in the protein–glucan complex obtained by precipitating the 24% NaOH extract at pH 6.0. This protein–glucan complex consisted of 39.3% polysaccharide and 49.4% protein. Its ¹³C- and ¹H-NMR data showed that the main glucan part of the complex was simple α-1,3-glucan chains.     

Mitochondrial DNA in Mon-Mon and Di-Mon Pairings of Pleurotus ostreatus

Based on enzymatically amplified regions of the mitochondrial DNA (mtDNA), stock-specific markers were obtained for two stocks of Pleurotus ostreatus. A length mutation was detected within a region encoding for DNA of the mitochondrial ribosomes. The inheritance of mtDNA was uniparental both in Mon-Mon and Di-Mon interstock pairings. Replacement of the recipient mtDNA to a large extent was finished 3 to 4 months after hyphal contact of paired mycelia. Dissolution of mitochondrial mosaics was somewhat faster in Di-Mon than in Mon-Mon pairings. As became evident in Di-Mon pairings, the spread of the dominating mtDNA is independent from nuclear migration, and these processes could go in opposite directions. While in compatible Di-Mon pairings two different types of nuclear background develop with respect to the mating type factors, only one certain mtDNA phenotype remained after dissolution of the mitochondrial mosaic. The spread of the mtDNA occurred at a distinctly slower rate than nuclear migration. Three weeks after hyphal contact the dominating mtDNA had not reached the growing edge of the recipient mycelium, whereas clamp formation was noted after 11 days at the latest. The data presented in this study indicate that no apparent correlation exists between mtDNA and nuclear background.     

RAC1 induces nuclear alterations through the LINC complex to enhance melanoma invasiveness

RHO GTPases are key regulators of the cytoskeletal architecture, which impact a broad range of biological processes in malignant cells including motility, invasion, and metastasis, thereby affecting tumor progression. One of the constraints during cell migration is the diameter of the pores through which cells pass. In this respect, the size and shape of the nucleus pose a major limitation. Therefore, enhanced nuclear plasticity can promote cell migration. Nuclear morphology is determined in part through the cytoskeleton, which connects to the nucleoskeleton through the Linker of Nucleoskeleton and Cytoskeleton (LINC) complex. Here, we unravel the role of RAC1 as an orchestrator of nuclear morphology in melanoma cells. We demonstrate that activated RAC1 promotes nuclear alterations through its effector PAK1 and the tubulin cytoskeleton, thereby enhancing migration and intravasation of melanoma cells. Disruption of the LINC complex prevented RAC1-induced nuclear alterations and the invasive properties of melanoma cells. Thus, RAC1 induces nuclear morphology alterations through microtubules and the LINC complex to promote an invasive phenotype in melanoma cells.     

Nuclear mRNA Export Requires Complex Formation between Mex67p and Mtr2p at the Nuclear Pores

We have identified between Mex67p and Mtr2p a complex which is essential for mRNA export. This complex, either isolated from yeast or assembled in Escherichia coli, can bind in vitro to RNA through Mex67p. In vivo, Mex67p requires Mtr2p for association with the nuclear pores, which can be abolished by mutating either MEX67 or MTR2. In all cases, detachment of Mex67p from the pores into the cytoplasm correlates with a strong inhibition of mRNA export. At the nuclear pores, Nup85p represents one of the targets with which the Mex67p-Mtr2p complex interacts. Thus, Mex67p and Mtr2p constitute a novel mRNA export complex which can bind to RNA via Mex67p and which interacts with nuclear pores via Mtr2p.     

COMPARATIVE ULTRASTRUCTURE OF THE PRIMARY NUCLEUS IN BRYOPSIS AND ACETABULARIA1,2

The fine structure of the primary nucleus in Bryopsis hypnoides was compared with that of the primary nucleus in Acetabularia calyculus and Batophora oerstedii. In Bryopsis the mature primary nucleus contains a peripheral reticulum composed of 4–6 layers of 200 A diameter fibrils. The nuclear membrane has numerous nuclear pores that are located adjacent to the openings in the layers of the reticulum. Each nuclear pore is constituted of peripheral globular elements and a central granule. Although pores of similar structure are present in the nuclear envelope of Acetabularia and Batophora, a fibrillar reticulum is absent.     

Parasexual analysis of common-A crosses in the basidiomycete Agrocybe aegerita

Summary Crosses were performed between homokaryons of Agrocybe aegerita having the same allele at the A incompatibility gene but different B alleles. Heterokaryotic mycelia originating from crosses between two complementary auxotrophs were characterized by their instability on complete medium and extensive anastomosis between hyphae. Diploid mycelia were selected by plating oidia recovered from these heterokaryons onto minimal medium. These mycelia were characterized by the production of larger oidia than those of homokaryons, the release of a brown pigment when growing on complete medium and extensive hyphal anastomoses. Diploids retained the two B incompatibility functions of their homokaryotic parents and gave rise to a diploid/haploid dikaryon when crossed with a compatible homokaryon. Nearly 1% of the oidia recovered from heterokaryons were diploid. These nuclear fusion frequencies as well as the production of brown pigments enabled the identification of diploid strains on complete medium. In this way, crosses between wild prototrophic strains were successfully performed. Somatic recombination was induced following the treatment of diploid mycelia with haploidizing compounds. Selection based on the inability of mycelia to produce the brown pigments on complete medium led to selection of strains homoallelic at the B locus.     

桦褐孔菌三萜类物质的提取与含量测定

以桦褐孔菌发酵菌丝体为材料,通过对溶剂乙醇（95%）、甲醇、乙酸乙酯、丙酮、异丙醇、正己烷和氯仿的提取效果比较表明,提取三萜类化合物的最佳溶剂为异丙醇,提取时间为24h,每个样品所需溶剂量（6mL）和菌丝体样品量（100mg）较少,并可同时对大量样品进行有效提取。以白桦脂醇为标准品,对香草醛-冰醋酸-高氯酸分光光度法进行评价,证明该方法简单快速、准确度高、试验误差小、重复性好。利用此方法对桦褐孔菌的野生菌核和发酵菌丝体内三萜类化合物含量进行测定,结果表明野生菌核（59.86mg/g）和发酵菌丝体（94.92mg/g）中都含有很高的三萜类化合物,且发酵菌丝体中三萜类化合物含量高于野生菌核。因此在桦褐孔菌产品开发中,发酵菌丝体可代替野生菌核进行大工业化生产。     

Nuclear and Non-nuclear Factors influencing Clamp Connection Formation in Coprinus disseminatus

Matings between sister single spore lines of Coprinus disseminatusshowed a cryptic tetrapolar pattern. The two groups of matingsthat resulted in formation of mycelia with clamp connections(apparent dikaryons) differed in rate of nuclear penetrationduring mating. In one group penetration occurred at rates comparablewith nuclear migration in other species and in the other groupit was often extensive but at a rate similar to, or less than,the dikaryon growth-rate. No differences were detected betweenthese two groups in stability, colony extension rate, frequencyof clamp connections, proportions of true clamp connectionsand pseudoclamps, or number of nuclei per hyphal tip cell. Cytological studies and the isolation of hyphal tips showedthat both groups of apparent dikaryons were heterokaryotic di-or trikaryons. The di- and trikaryotic conditions co-existedin the same mycelium, but adjacent cells of individual branchingsystems usually contained equal numbers of nuclei. Within apparentdikaryons the number and kinds of nuclei per cell were similarin hyphae with clamp connections and those with simple septa.Treatments that prevented clamp connection formation did notalter the nuclear status of most of the hyphae. Irregularities in nuclear distribution were infrequent and mostwere associated with pseudoclamps. Forty per cent of nodes withprobable pseudoclamps yielded homokaryotic branches, which wereof either constituent mating type. There was some indicationthat irregularities in nuclear distribution could also occurduring divisions associated with simple septa.     

Mechanisms and Signals for the Nuclear Import of Proteins

In eukaryotes, the nuclear membrane provides a physical barrier to the passive diffusion of macromolecules from and into the cytoplasm. Nucleocytoplasmic traffic occurs through highly specialized structures known as nuclear pores, and involves the participation of a special class of transport proteins. Active transport across the nuclear pores is an energy-dependent process that relies on the activity of Ran-GTPases both in the nuclear and cytoplasmic compartments. Nuclear import of proteins is an essential step in regulating gene expression and the replication cycle of several viruses. In this review, the key mechanisms, pathways, and models underlying the transport of proteins across nuclear pores are analysed.Key Words: Nuclear pore complex, nuclear localization signal, importin, nuclear transport.     

TIME SEQUENCE OF NUCLEAR PORE FORMATION IN PHYTOHEMAGGLUTININ-STIMULATED LYMPHOCYTES AND IN HELA CELLS DURING THE CELL CYCLE

The time sequence of nuclear pore frequency changes was determined for phytohemagglutinin (PHA)-stimulated human lymphocytes and for HeLa S-3 cells during the cell cycle. The number of nuclear pores/nucleus was calculated from the experimentally determined values of nuclear pores/µ² and the nuclear surface. In the lymphocyte system the number of pores/nucleus approximately doubles during the 48 hr after PHA stimulation. The increase in pore frequency is biphasic and the first increase seems to be related to an increase in the rate of protein synthesis. The second increase in pores/nucleus appears to be correlated with the onset of DNA synthesis. In the HeLa cell system, we could also observe a biphasic change in pore formation. Nuclear pores are formed at the highest rate during the first hour after mitosis. A second increase in the rate of pore formation corresponds in time with an increase in the rate of nuclear acidic protein synthesis shortly before S phase. The total number of nuclear pores in HeLa cells doubles from ~2000 in G₁ to ~4000 at the end of the cell cycle. The doubling of the nuclear volume and the number of nuclear pores might be correlated to the doubling of DNA content. Another correspondence with the nuclear pore number in S phase is found in the number of simultaneously replicating replication sites. This number may be fortuitous but leads to the rather speculative possibility that the nuclear pore might be the site of initiation and/or replication of DNA as well as the site of nucleocytoplasmic exchange. That is, the nuclear pore complex may have multiple functions.     

A strategy for screening microbial strains with lipolytic specificity toward monoacylglycerols

For easy obtaining the microorganisms with lipolytic specificity toward monoacylglycerols, we developed a simple and effective method to isolate the objective strains. This method employed a nile-blue agar-plate culture containing mono- and tri- acylglycerols for microorganism screening and selected the desired microorganisms by analysis of free fatty acid contents in lipid extracts obtained from culture broth. Using this strategy, we successfully isolated one mold strain with superior lipolytic ability for the hydrolysis of monoacylglycerols. The mold was identified and designated as Paecilomyces nostocoides NTU-FC-LP01. The lyophilized mycelia of the isolated mold used as a biocatalyst showed high specificity toward monoacylglycerols rather than di- and tri- acylglycerols. Furthermore, the lyophilized mycelia catalyzed the monoolein synthesis through the direct esterification of oleic acid and glycerol. It indicated that the lyophilized mycelia of the present P. nostocoides NTU-FC-LP01 could be used as a natural immobilized biocatalyst for the glycerol/oleic acid esterification to produce monoolein.