Mitotic characteristics of heterophase homokaryons

Heterophase homokaryons were obtained by fusion of the Chinese hamster culture cells. About 94% of these homokaryons were divided by the multipolar mitosis. Chromosome sets arising from different nuclei were found to keep segregations in common metaphase plates both in the multipolar and bipolar mitoses.     

Expression of Proteins and Glycoproteins Encoded by the Haploid Nuclei in the Dikaryotic State in the Basidiomycete Agrocybe aegerita

The total proteins and concanavalin A-binding glycoproteins of the cultivated mushroom Agrocybe aegerita were studied in homokaryotic siblings and in dikaryotic strains. The glycoproteins exhibited considerable variability compared with the proteins; the genetic diversity detected in homokaryons in the glycoprotein analysis was 30-fold higher than the genetic diversity revealed by protein analysis, and the glycoprotein patterns could be used to characterize individual genotypes. We found that the expression of glycoproteins in haploid nuclei was significantly asymmetric when the nuclei were paired in dikaryons. The expression levels of the two component nuclei depended on their genotypes, and each haploid nucleus was characterized by its level of expression. Furthermore, some specific glycoproteins that were not detected in all of the homokaryons were newly synthesized in the dikaryotic strains. Among these was a glycoprotein designated gpAa-65, which was identified in all of the dikaryotic strains and appeared to be a good molecular marker of the dikaryotic state.     

Comparative measurements of membrane potentials with microelectrodes and voltage-sensitive dyes

The usefulness of a new voltage-sensitive fluorescent dye, the membrane permeant negatively charged oxonol dye diBA-C4-(3)-, was evaluated by measuring the membrane potentials of BICR/M1R-k and L cells with glass microelectrodes and simultaneously recording the fluorescence of the stained cells. The membrane potential of BICR/M1R-k cells was varied between -25 mV and -90 mV by changing the bicarbonate concentration in the medium or by voltage clamping. To avoid any interference by the inserted electrodes with the fluorescence measurement of the cytoplasm, the cells were fused by polyethyleneglycol to form giant cells (homokaryons). These homokaryons also allowed penetration by two glass microelectrodes without causing a serious leakage of the plasma membrane. The slow responding dye diBA-C4-(3)- had a fluorescence response of about 1% per mV. Mathematical analysis of the fluorescence changes after voltage clamping revealed a first-order reaction with a rate constant between 0.1 min-1 and 0.8 min-1, depending on the cell size which was determined by the number of nuclei per homokaryon. A model for the mechanism of the fluorescence changes is proposed.     

表型与亲本不易区分的茯苓同核体菌株新类型

茯苓Wolfiporia hoelen是我国传统中药材之一,也是一种食药兼用的大型真菌,目前已规模化栽培,但由于其交配系统一直不明确,影响了种质改良。前期我们发现了茯苓的同核体,明确了茯苓的交配系统和生活史,并建立了以培养特性和分子标记区分同核体的方法,但未明确是否适用于茯苓种群的不同个体。在对多个菌株的研究中,发现了同核体表型与亲本不易区分的茯苓菌株。本研究主要以来自日本的菌株775 (NBRC 30628)为亲本,对其同核体菌株进行收集鉴定,并对同核体菌株的培养特性、交配现象和杂交等进行了研究。此类菌株的同核体菌株可通过与亲本对峙培养进行鉴定,但菌丝生长、菌落形态和吃料速度等与亲本没有显著差异,不同交配型的同核体之间交配时没有明显的交配现象。rpb2杂合位点标记可以用于鉴定该类型同核体菌株,且能验证是否交配。该类型同核体与之前发现的同核体类型之间可以进行杂交,杂交菌株可与两亲本都产生拮抗现象。该发现补充了之前建立的茯苓同核体鉴定方法,加深了对茯苓物种群体的了解,同时丰富了茯苓的育种资源。     

Genetic evidence for somatic haploidization in developing fruit bodies of Armillaria tabescens

Armillaria spp. have vegetative hyphae with diploid uninucleate cells, but the fruit bodies of many species contain clamped dikaryotic hyphae. Earlier observations suggest that somatic haploidization takes place in developing fruit bodies. To verify this, a uninucleate diploid cell was isolated from each of the 49 mating combinations between single-spore isolates of Armillaria tabescens and they were fruited. Twenty-four isolates produced fruit bodies with at least a partially dikaryotic subhymenium. Dikaryotic hyphae were isolated from fruit-body primordia and homokaryons were obtained by micromanipulation or by protoplasting. Approximately half of the isolates proved to represent recombinant mating types in respect to parent homokaryons, and most of them contained recombinant haploid DNA, based on random-amplified microsatellite markers. The results show that the nuclei in dikaryotic hyphae found in fruit bodies result from somatic haploidization. The mechanism of haploidization remains unclear.     

Genetic Variability in Pectic Enzymes of Rhizoctonia solani Isolates Causing Bare-patch Disease of Cereals

Field isolates of Rhizoctonia solani obtained from three discrete bare patches in a wheat field in Western Australia were characterized by pectic zymogram grouping. The genetic background of pectic enzymes was analysed by comparing the zymograms of asexual homokaryons and sexual progenies derived from field isolates. The 170 field isolates obtained from the field site produced indistinguishable pectic zymograms. However, variations among field isolates of the same zymogram group were detected, on the basis of zymograms of their resultant protoplast-regenerated cultures. Asexual sibling homokaryons derived from each of the field isolates were heterogeneous for their pectic enzymes. Homokaryons with a common heterokary on incompatibility factor, obtained from a field isolates were homogeneous for pectic enzymes. Basidiospore progenies of a field isolate segregated widely in pectic zymograms. It appeared that the expression of pectic enzymes by field isolates involved multiple genetic factors. The variation of zymograms among homokaryotic strains suggests that each field isolate of R. solani contains two types of nuclei, although cells of vegetative hyphae are multinucleate.     

Nuclear selection in oidium formation from dikaryotic mycelia ofFlammulina velutipes

The effect of nuclear dominance in monokaryotic oidium formation from dikaryotic mycelia in a tetrapolar basidiomycete,Flammulina velutipes, was examined. A total of 46 monokaryotic stocks were used to produce 194 hybrid dikaryotic stocks by crossing. The proportion of homokaryons among the oidium isolates from dikaryotic mycelia was over 95%. The staining of nuclei of oidia with propidium iodide showed that over 90% of oidia were monokaryotic and suggested that these oidia had single haploid nuclei at the G1 stage. The monokaryotic oidium isolates from hybrid dikaryons were backrossed to parental monokaryotic stocks. Although most of the monokaryotic oidium isolates (except for those from 17 hybrid dikaryons from a total of 194 test stocks) showed nuclear types similar to only one of the parental stocks, the process seems to produce essentially the split nuclear type composition. Therefore, the monokaryotization in oidium formation from dikaryotic mycelia essentially involves the process of nuclear selection. The two separate results of hierarchies of relative dominance among two nuclei of the parental dikaryons in the monokaryotic oidium formation by grouping with incompatibility factor compositions were determined. Only a few discrepancies were found in the hierarchies between the two specific nuclear compositions of hybrid dikaryons.     

Analysis of Inbreeding Depression in Agaricus Bisporus

Inbreeding depression was observed in the commercial button mushroom, Agaricus bisporus, by examining two laboratory populations. The outbred population consisted of 20 compatible pairings, 10 homokaryons with each of the homokaryons Ag1-1 and Ag89-65. The inbred population consisted of 104 backcrosses (among which 52 were expected to be sexually compatible) obtained from the pairings of two progenitor homokaryons, Ag1-1 and Ag89-65, with 52 progeny homokaryons derived from the mating between Ag1-1 and Ag89-65. The eight fitness components examined for these two populations were successful matings as identified by the analysis of restriction fragment length polymorphisms, positive mycelial interaction in these successful matings, heterokaryon growth rate, primordium formation by the successful matings, fertile fruiting body formation, time to first break, average number of fruiting bodies per square foot, and average weight per fruiting body. The outcrossed population showed a significant advantage over the inbred population in three of eight fitness components. Two pairs of traits were significantly correlated. The multiplicative fitness ratio of the inbred to the outcrossed population was 0.18. The relevance of inbreeding depression to the evolution of fungal mating systems and to mushroom breeding is discussed.     

Fusion Experiments of Isolated Generative Cells in Several Angiosperm Species

The generative cells used for fusion experiments were isolated from pollen grains of Zephyranthes candida and Lycoris radiata by “2-step osmotic shock” and from those of Hippeastrum vittata, Hemerocallis minor and Iris tectorum by “weak enzyme treatment” as reported previously. Using PEG method, fusions have been successfully induced between generative cells of the same species mentioned above, between generative cells of Z. candida and L. radiata, between generative cells and petal protoplasts in L. radiata, and between generative cells of L. radiata and hypocotyl protoplasts of Brassica napus. In all cases either homokaryons or heterokaryons could be obtained. Fusion of nuclei was observed sometimes in homokaryons of generative cells in L. radiata. The generative nuclei in fusion products could be well identified by labelling the generative cells before fusion with DAPI. FDA test demonstrated that most of the fusion products were viable. Factors affecting fusion efficiency including cell density, PEG concentration, duration of PEG treatment and effect of calcium ions were studied in fusion of generative cells in Z. candida. Our experiments indicate that isolated generative cells are likely to be deprived of cell wails and may be regarded as a special kind of protoplasts for direct fusion experiments.     

Homokaryons are more combative than heterokaryons of Hericium coralloides

The homokaryotic stage of the basidiomycete lifecycle is generally considered to be short lived, although there is little experimental evidence relating to their longevity in the field. The vast majority of studies on basidiomycete ecology have used only heterokaryons. The few investigations comparing related homokaryons and heterokaryons have revealed no overall trend in differences of extension rate, wood decay or competitive ability. For a rare species the homokaryotic phase may be of greater importance than in common species as it is likely to last longer. Hericium coralloides, a rare wood decay basidiomycete, was used to investigate differences between homokaryons and heterokaryons in terms of extension rate and combative ability. Fifteen homokaryons from three fruit bodies and five heterokaryons (obtained by fruit body tissue isolation) were compared at 5–35 °C on malt agar for extension rate, and paired against heterokaryons of 13 wood decay species to assess combative ability. Homokaryons were paired to create ten artificial heterokaryons whose extension rate at 10 and 20 °C was compared to parental rates. There were some significant differences in extension rates between homokaryons and natural heterokaryons, between homokaryons and heterokaryons created artificially from homokaryons, and between homokaryons from different fruit bodies, but no consistent trends. Homokaryons proved more combative than heterokaryons, which was assessed quantitatively as well as qualitatively using a scoring system for outcome of each pairing. Results are discussed in relation to previous findings and in an ecological context.     

Variations in culture morphology and pathogenicity among protoplast-regenerated strains of Rhizoctonia solani

Abstract Protoplast-regenerated cultures derived from mycelia of cereal-infecting field isolates of Rhizoctonia solani exhibited major variations in cultural morphology and in pathogenicity. Each field isplate yielded three of four distinct morphological types of protoplast cultures. The presence of the new morphological phenotypes was attributed to the selection of homokaryons arising from protoplasts with single nuclei. Highly pathogenic field isolates produced protoplast cultures with higher virulence than those from weakly virulent pathogenic isolates, and homokaryotic strains were generally less pathogenic than the parental field isolate.     

Fibronectin expression is determined by the genotype of the transformed parental cells in heterokaryons between normal and transformed fibroblasts

The expression of fibronectin, a cell surface-associated transformation-sensitive glycoprotein, was studied in hetero- and homokaryons of normal and SV40-transformed human fibroblasts. In immunofluorescence, fibroblast homokaryons had an intense surface-associated and intracelluar fibronectin fluorescence similar to that of normal fibroblasts. Transformed cells and their homokaryons had a minimal surface-associated and a weak intracellular fibronectin fluorescence. In heterokaryons formed between transformed and normal fibroblasts, the expression of fibronectin fell within 24 h to the level of the transformed cell homokaryons. The change was detectable already at 3 h after fusion and was gene-dose dependent. These results show that the transformed genotype determines fibronectin expression in the heterokaryons.     

Variability in Mating Behaviour and Culture Appearance of Lenzites trabea

Twenty-three homokaryons of Lenzites trabea (Pers.) Fr. wereproduced, mainly by chemical monokaryotization, from dikaryonsisolated from widely separated regions throughout the world.These homokaryons possessed 18 different mating factor alleles,so an estimate was made of the total number of alleles in theworld population of 51, 45, or 36, depending on the method ofestimation. Some variability in the production of clamp connectionsduring mating was noted. It was also found that the appearanceof the cultures varied considerably, especially that of thehomokaryons. A brief study was made of this variability on severalculture media, and the appearance of dikaryons and their constituenthomokaryons was compared on malt agar.     

Analysis of lethal events induced by ultraviolet in a heterokaryon of Neurospora.

Summary The relative frequencies of heterokaryons and the two kinds of homokaryons have been scored among colonies from conidia harvested from a heterokaryon and treated with UV, in order to determine which kinds of lethal mutations were induced. Recessive lethal mutations were scored directly. The pattern of surviving types indicated that recessive lethals and mitotic lethals (events destroying whole nuclei) occurred with similar frequencies. But the absolute frequency of these mutations was not sufficient to account for the observed kill, suggesting that dominant lethals and/or cytoplasmic lethals were also induced at a similar rate.     

Dominant versus recessive behavior of a cold- and a heat-sensitive mammalian cell cycle variant in heterokaryons

A heat-sensitive (hs, arrested at 39.5 degrees C, termed 21-Ta) and a cold-sensitive (cs, arrested at 33 degrees C, termed 21-Fb) clonal cell cycle variant were isolated from the same clone of the P-815 murine mastocytoma line. At the respective nonpermissive temperatures, both the hs and the cs variant were reversibly arrested in G1 phase, and numbers of cells forming colonies upon reincubation at the permissive temperature remained nearly constant for at least 6 days. Cells arrested in G1 by incubation at the respective nonpermissive temperatures were fused to cells of another P-815 clone (31-S) that had been arrested by serum deprivation. Upon reincubation in medium containing 10% serum for 48 h at 39.5 degrees C, 21-Ta x 31-S heterokaryons, similar to 31-S x 31-S homokaryons, entered the S phase, whereas at 33 degrees C, 21-Fb x 31-S heterokaryons, similar to 21-Fb x 21-Fb homokaryons, remained arrested in G1, indicating a recessive expression of the hs and a dominant expression of the cs phenotype.     

Nuclear migration and mitochondrial inheritance in the mushroom agaricus bitorquis

Mitochondrial (mt) DNA restriction fragment length polymorphisms (RFLPs) were used as genetic markers for following mitochondrial inheritance in the mushroom Agaricus bitorquis. In many basidiomycetes, bilateral nuclear migration between paired homokaryotic mycelia gives rise to two discrete dikaryons which have identical nuclei but different cytoplasms. Although nuclear migration is rare in A. bitorquis, unidirectional nuclear migration occurred when a nuclear donating strain (8-1), was paired with a nuclear recipient strain (34-2). The dikaryon recovered over the nuclear recipient mate (Dik D) contained nuclei from both parents but only mitochondria from the recipient mate; thus nuclei of 8-1, but not mitochondria, migrated through the resident hyphae of 34-2 following hyphal anastomosis. The two mitochondrial types present in a dikaryon recovered at the junction of the two cultures (Dik A) segregated during vegetative growth. Dikaryotic cells having the 34-2 mitochondrial type grew faster than cells with the 8-1 mitochondrial type. Fruitbodies, derived from a mixed population of cells having the same nuclear components but different cytoplasms, were chimeric for mitochondrial type. The transmission of mitochondria was biased in favor of the 8-1 type in the spore progeny of the chimeric fruitbody. Protoplasts of dikaryon (Dik D), which contained both nuclear types but only the 34-2 mitochondrial type, were regenerated and homokaryons containing the 8-1 nuclear type and the 34-2 mitochondrial type were recovered.     

Genetic evidence for nonrandom sorting of mitochondria in the basidiomycete Agrocybe aegerita.

We studied mitochondrial transmission in the homobasidiomycete Agrocybe aegerita during plasmogamy, vegetative growth, and basidiocarp differentiation. Plasmogamy between homokaryons from progeny of three wild-type strains resulted in bidirectional nuclear migration, and the dikaryotization speed was dependent on the nuclear genotype of the recipient homokaryon. Little mitochondrial migration accompanied the nuclear migration. A total of 75% of the dikaryons from the fusion lines had both parental mitochondrial haplotypes (mixed dikaryons), and 25% had only a single haplotype (homoplasmic dikaryons); with some matings, there was a strong bias in favor of one parental haplotype. We demonstrated the heteroplasmic nature of mixed dikaryons by (i) isolating and subculturing apical cells in micromanipulation experiments and (ii) identifying recombinant mitochondrial genomes. This heteroplasmy is consistent with the previously reported suggestion that there is recombination between mitochondrial alleles in A. aegerita. Conversion of heteroplasmons into homoplasmons occurred (i) during long-term storage, (ii) in mycelia regenerated from isolated apical cells, and (iii) during basidiocarp differentiation. Homokaryons that readily accepted foreign nuclei were the most efficient homokaryons in maintaining their mitochondrial haplotype during plasmogamy, long-term storage, and basidiocarp differentiation. This suggests that the mechanism responsible for the nonrandom retention or elimination of a given haplotype may be related to the nuclear genotype or the mitochondrial haplotype or both.     

Crosses among Homokaryons from Commercial and Wild-Collected Strains of the Mushroom Agaricus brunnescens (= A. bisporus)

A new technique for the production of hybrid strains of the cultivated mushroom Agaricus brunnescens is described. Homokaryons were recovered from regenerated protoplasts obtained from several heterokaryotic strains. A total of 16 novel hybrids were produced in 63 attempted crosses between paired homokaryons. Recovery of both homokaryons and hybrids was verified by analysis of restriction fragment length polymorphisms. Three of four hybrids fruited in small-scale tests, further confirming that the isolates were true hybrids. Colony morphology alone was found to be a poor indicator of hybrid status. In two instances, three homokaryons crossed successfully in all combinations, suggesting that there are at least three alleles at the putative mating-type locus. Crosses between homokaryons from commercial and wild-collected isolates indicated that these strains belong to the same biological species.     

A shift in nuclear state as the result of natural interspecific hybridization between two North American taxa of the basidiomycete complex Heterobasidion

A natural first generation hybrid fungus shows interspecific heterozygosity. The nuclear condition of a rare natural hybrid between two taxa of the Heterobasidion complex is investigated. Heterobasidion species are known to be either homokaryotic (haploid) or heterokaryotic (n+n), but heterokaryons are made up of both homokaryotic and heterokaryotic sectors. The natural hybrid appears to be either a heterokaryon undergoing a primary homothallic phase or a diploid with limited ability to exchange nuclei when mated with homokaryons. The natural hybrid is stable and long lived, suggesting hybridization may play an important role in the evolutionary history of this fungal complex.     

Cell cycle re-entry of quiescent mammalian nuclei following heterokaryon formation

When 3T3 mouse fibroblasts are made quiescent by serum deprivation and are then fused with tsAF8 hamster fibroblasts synchronized by a combination of high temperature block and hydroxyurea, the nuclei of binucleated heterokaryons which are formed enter S phase asynchronously in media containing low levels of serum. The tsAF8 nuclei of these biphasic heterokaryons enter S phase shortly after fusion, as do the tsAFS nuclei of homokaryons in the same culture. In contrast, the nuclei of the biphasic heterokaryons which have been contributed by quiescent 3T3 enter S phase only after a lag following fusion. This suggests that the quiescent nucleus within the heterokaryon is stimulated by factor(s) from the more advanced cell to re-enter the cell cycle in the absence of serum. In contrast to factors which induce the immediate synthesis of DNA, these factors may be those responsible for the transition of a cell from a non-proliferating to a proliferating state.