产黄青霉ds RNA病毒通过原生质体融合的转移

将国内青霉素产生菌(Penicillium chrysogenum)的黄孢子系统及绿孢子(包括淡绿,灰绿)系统的十多个菌株,经过病毒提取、电镜观察、奥氏免疫双扩散、凝胶电泳及放射免疫测定,证明黄孢子系统的菌株含有不同滴定度的、直径40nm的球形病毒,而绿孢子系统中检查不出病毒。从营养要求、孢子颜色不同的带病毒和无病毒菌体中分离原生质体,进行不同组合的原生质体的融合杂交,获得营养互补融合的异核体。异核体1中,病毒通过胞质融合转移到原来无病毒的灰绿孢子菌株及细胞核融合后的杂合二倍体中。灰绿孢子的病毒量接近二倍体的1/3。二倍体菌落生长稳定,低温保存二年后经0.01—0.02M对氟苯丙氨酸(PFA)诱发和分离,产生亲本类型的分离子,分离子及二倍体仍然含有病毒。异核体2作亲本性分离,黄孢子仍有病毒,淡绿孢子及细胞核融合后产生的二倍体均无病毒,表明非感染性为显性。此种淡绿孢子的突变体中存在非感病菌系,它不支持病毒的复制。提取各杂交组二倍体内的病毒所特有的dsRNA时,可看出dsRNA的存在和病毒的存在一致。多数杂合二倍体的青霉素产量比亲本高。     

Roles for receptors, pheromones, G proteins, and mating type genes during sexual reproduction in Neurospora crassa

Here we characterize the relationship between the PRE-2 pheromone receptor and its ligand, CCG-4, and the general requirements for receptors, pheromones, G proteins, and mating type genes during fusion of opposite mating-type cells and sexual sporulation in the multicellular fungus Neurospora crassa. PRE-2 is highly expressed in mat a cells and is localized in male and female reproductive structures. Δpre-2 mat a females do not respond chemotropically to mat A males (conidia) or form mature fruiting bodies (perithecia) or meiotic progeny (ascospores). Strains with swapped identity due to heterologous expression of pre-2 or ccg-4 behave normally in crosses with opposite mating-type strains. Coexpression of pre-2 and ccg-4 in the mat A background leads to self-attraction and development of barren perithecia without ascospores. Further perithecial development is achieved by inactivation of Sad-1, a gene required for meiotic gene silencing. Findings from studies involving forced heterokaryons of opposite mating-type strains show that presence of one receptor and its compatible pheromone is necessary and sufficient for perithecial development and ascospore production. Taken together, the results demonstrate that although receptors and pheromones control sexual identity, the mating-type genes (mat A and mat a) must be in two different nuclei to allow meiosis and sexual sporulation to occur.     

Two Cell Division Cycle Mutants of SACCHAROMYCES CEREVISIAE Are Defective in Transmission of Mitochondria to Zygotes

Mutations in CDC genes of S. cerevisiae disrupt the cell cycle at specific stages. The experiments reported here demonstrate that two CDC genes, CDC5 and CDC27, are necessary for mitochondrial segregation as well as for nuclear division. The defect in the transmission of mitochondria was revealed by the examination of uninucleate and binucleate progeny of transient heterokaryons generated by using the kar1-1 mutation that disrupts nuclear fusion. One of the parents lacked mitochondrial DNA (ρ⁰) whereas the other parent had functional mitochondria (ρ⁺). When the parents of the heterokaryon were both wild-type (CDC), nearly all progeny received mitochondria at 21° and at 34°. Thirty-four of the 36 cdc mutations tested had no defect in transmission of mitochondria to zygotic progeny in crosses in which one parent was a cdc mutant and the other parent was not (CDC). However, the cdc5 and cdc27 mutations prevented the transmission of mitochondria to cdc progeny at 34° but not at 21°; CDC progeny received mitochondria at either temperature. This defect was observed in crosses of cdc5 or cdc27 by wild-type cells regardless of which parent donated mitochondria to the zygote. The defect in mitochondrial transmission cosegregated in meiotic tetrads with the defect in mitosis demonstrating that both are likely to be caused by the same temperature-sensitive mutation. These results indicate that the CDC5 and CDC27 gene products are essential in two motility-related processes: mitochondrial movement from the zygote to the progeny and in mitosis.—Furthermore, the results suggest that the function performed by the CDC5 and CDC27 gene products for mitochondrial transmission differ in some fundamental way from the function performed for mitosis. The function necessary for mitosis can be supplied to the cdc5 (or cdc27) nucleus by the CDC5 (or CDC27) nucleus in the same heterokaryon but the function necessary for mitochondrial transmission cannot. Perhaps the function needed for mitochondrial transmission must be performed in the cell cycle preceding the actual segregation of mitochondria whereas the function needed for nuclear segregation can be performed at the time that mitosis occurs.     

Heterokaryon Analysis of the Genetic Control of Pigment Synthesis in Chick Embryo Melanocytes

The genetic control of pigmentation was analyzed using five unlinked mutants, namely, c, pk, Bl, e^y and I. Each mutant blocks or reduces pigmentation. Chick melanocyte cultures of each mutant type were fused to produce all ten possible pair combinations of nondividing heterokaryons. Heterokaryons were identified autoradiographically. (One partner in each pair was labeled with ³H-thymidine.) Crosses produced comparable pairs of double heterozygotes that were analyzed in vivo and in vitro. Heterokaryon pairs were compared to their corresponding double heterozygotes.—Some combinations showed complementation and produced wild-type pigment. Others showed noncomplementation having little or no pigment. Double heterozygotes complemented each other except in the cases involving the dominant mutant, I. Four heterokaryon pairs gave different results from their corresponding double heterozygotes. The pk-Bl and pk-e^y combinations failed to complement as heterokaryons but did complement as double heterozygotes. On the other hand, the I-c and I-Bl combinations complemented as heterokaryons but not as double heterozygotes. Based on these differences it is hypothesized that the pk and I loci are nuclearly restricted regulatory elements. Examples in the literature from other systems are cited to support such a hypothesis.     

Mutations of the a Mating-Type Gene in NEUROSPORA CRASSA

In Neurospora, the mating-type locus controls both mating ( A + a is fertile) and heterokaryosis (A + a is incompatible). The two alleles appear stable: no novel fertility reactions have ever been reported, and attempts to separate fertility and heterokaryon incompatibility functions by recombination have been unsuccessful. In the present approach the locus was studied through a mutational analysis of heterokaryon incompatibility function. A selection system was used that detects vigorous (A + a) heterokaryotic colonies against a background of inhibited growth. Twenty-five mutants of an a strain were produced following mutagenic treatment with UV and NG: 15 were viable as homokaryons and 10 were not. All but one were infertile, but most showed an abortive mating reaction involving the production of barren, well-developed perithecia with A and (surprisingly) a testers. None of the mutants complement each other to restore fertility. Seven mutants have been mapped to the mating-type locus region of chromosome 1. Restoration of fertility was used to detect revertants, and these were found in five out of the eight mutants tested. (A dose response was observed). In four cases incompatibility was fully restored and in one case it was not.—The results suggest two positive actions of the locus when in heterozygous (A/a) combination (the stimulation of some stage of ascus production and the inhibition of vegetative heterokaryosis), and one positive action in homozygous combination (the production of a perithecial inhibitor).     

Mating-type heterokaryosis and selfing in Cryphonectria parasitica

Selfing in the chestnut blight fungus, Cryphonectria parasitica, occurs by two different genetic mechanisms. Most self-fertile isolates of C. parasitica are heterokaryotic for mating type, and the progeny from selfing segregate for mating type. Further, we resolved mating-type (MAT) heterokaryons into homokaryons of both mating types by isolating uninucleate asexual spores (conidia). However, because ascospore progeny, with rare exceptions, are not MAT heterokaryons, C. parasitica must lack a regular mechanism to maintain heterokaryosis by selfing. We hypothesize that heterokaryon formation may occur either because of recurrent biparental inbreeding, or by mating-type switching, possibly one involving some kind of parasexual process. The second mechanism found for selfing in C. parasitica occurred less frequently. Three single-conidial isolates (MAT-1 and MAT-2) selfed and produced progeny that did not segregate for mating type. It is currently not known if meiosis occurs during ascospore formation by this mechanism.     

Intraspecific hybridization of Trichoderma reesei by protoplast fusion

Protoplasts obtained from mycelia of a single auxotrophic mutant of Trichoderma reesei QM 9414 were fused with those of T. reesei QM 9136 in the presence of 0.5 M glycine-NaOH buffer, pH 7.5, containing 0.05 M CaCl₂ · 2H₂O and 35% polyethylene glycol 4,000. The regeneration frequency of these protoplasts was 8.9–12.0% on a solid culture medium with soft agar overlay. The fused protoplasts successfully formed heterokaryons showing 3.33% of the fusion frequency. A heterozygous diploid was obtained from conidia of the heterokaryon by treatment with 0.1% d-camphor. The diploid showed a 1.9 fold DNA content per conidial nucleus compared to T. reesei QM 9414. The frequency of diploid formation was about 1.9 × 10⁻⁴ per conidium. Cellulase activities, such as filter paper degrading and CM-cellulose and Avicel saccharifying activities, and the xylanase activity of the diploid showed intermediate values between those of T. reesei QM 9414 and T. reesei QM 9136. However, the β-glucosidase, β-1,3-glucanase and chitinase activities of the diploid increased to levels equal to on above those of T. reesei QM 9414 and T. reesei QM 9136. The existence of a parasexual cycle of T. reesei and the possibility of its application to enhanced enzyme productivity were confirmed using the protoplast fusion technique.     

The Use of Duplication-Generating Rearrangements for Studying Heterokaryon Incompatibility Genes in Neurospora

Heterokaryon (vegetative) incompatibility, governing the fusion of somatic hyphal filaments to form stable heterokaryons, is of interest because of its widespread occurrence in fungi and its bearing on cellular recognition. Conventional investigations of the genetic basis of heterokaryon incompatibility in N. crassa are difficult because in commonly used stocks differences are present at several het loci, all with similar incompatibility phenotypes. This difficulty is overcome by using duplications (partial diploids) that are unlikely to contain more than one het locus. A phenotypically expressed incompatibility reaction occurs when unlike het alleles are present within the same somatic nucleus, and this parallels the heterokaryon incompatibility reaction that occurs when unlike alleles in different haploid nuclei are introduced into the same somatic hypha by mycelial fusion.—Nontandem duplications were used to confirm that the incompatibility reactions in heterokaryons and in duplications are alternate expressions of the same genes. This was demonstrated for three loci which had previously been established by conventional heterokaryon tests—het-e, het-c and mt. These were each obtained in duplications as recombinant meiotic segregants from crosses heterozygous for duplication-generating chromosome rearrangements. The particular method of producing the duplications is irrelevant so long as the incompatibility alleles are heterozygous.—The duplication technique has made it possible to determine easily the het-e and het-c genotypes of numerous laboratory and wild strains of unknown constitution. In laboratory strains both loci are represented simply by two alleles. Analysis of het-c is more complicated in some wild strains, where differences have been demonstrated at one or more additional het loci within the duplication used and multiple allelism is also possible.—The results show that the duplication method can be used to identify and map additional vegetative incompatibility loci, without the necessity of heterokaryon tests.     

Complementation Analysis of Linked Circadian Clock Mutants of NEUROSPORA CRASSA

A fourth mutant of Neurospora crassa, designated frq-4, has been isolated in which the period length of the circadian conidiation rhythm is shortened to 19.3 ± 0.3 hours. This mutant is tightly linked to the three previously isolated frq mutants, and all four map to the right arm of linkage group VII about 10 map units from the centromere. Complementation tests suggest, but do not prove, that all four mutations are allelic, since each of the four mutants is co-dominant with the frq⁺ allele—i.e., heterokaryons have period lengths intermediate between the mutant and wild-type—and since heterokaryons between pairs of mutants also have period lengths intermediate between those of the two mutants.     

Temperature and Sporulation of Aquatic Hyphomycetes

Temperature appears to be an important factor affecting the occurrence and distribution of aquatic hyphomycetes, the dominant leaf litter-decomposing fungi in streams. We compared conidium production by eight species of aquatic hyphomycetes grown on yellow poplar leaves in stream-simulating microcosms at three temperatures (15, 20, and 25°C). The greatest conidium production occurred at 15°C for one species, 20°C for two species, and 25°C for two species. Two species produced similar numbers of conidia at 20 and 25°C, and one species produced similar numbers of conidia at all three temperatures. Linear growth rates were determined on malt extract agar. Six species had the same pattern of temperature responses for growth on malt extract agar as for sporulation on leaves, as shown by the positive correlations between the two parameters at the three temperatures. The species examined also exhibited differences in number of conidia produced from a similar amount of leaf material at a given temperature. These differences appeared to be due primarily to differences in individual conidium mass (determined by weighing conidia produced from cultures), as shown by the relationship of the type Y = k/X (r² = 0.96), where Y is the number of conidia produced, X is the individual conidium mass in milligrams, and k is a constant empirically determined to be 2.11. This finding supports the hypothesis that aquatic hyphomycetes allocate similar amounts of their resources to reproduction but vary with respect how these resources are partitioned into reproductive units (conidia).     

HETEROKARYOSIS AND PARASEXUALITY IN THE FUNGUS ASCOCHYTA IMPERFECTA

The object of this investigation was to discover whether heterokaryosis and parasexuality occur in the imperfect fungus Ascochyta imperfecta. Both phenomena have been observed. The wild type of A. imperfecta grows on a minimal medium containing only salts plus a carbon source. Auxotrophic and morphological mutants have been isolated after treatment with ultraviolet light. When 2 different mutant auxotrophs are inoculated together onto minimal medium, colonies are consistently formed. These colonies might be due, a priori, to back-mutation, diploidy, syntrophism or heterokaryosis. Back-mutation and diploidy have been eliminated, since no back-mutant nuclei have been isolated from any heterokaryon, and since the frequency of diploid nuclei is very low. The combination is primarily syntrophic (only 2% heterokaryotic hyphal tips) when the nicotinamide mutant is one component. The combination is primarily heterokaryotic (over 50% heterokaryotic hyphal tips) when both components are auxotrophs for amino acids. From the heterokaryotic hyphal tips, the 2 unaltered nuclear components have been isolated. Heterozygous diploid nuclei (4.2 X 10^−-7 per haploid nucleus) can be isolated from heterokaryons by plating, onto minimal medium, the primarily uninucleate conidia from a heterokaryon of 2 auxotrophs. The resulting colonies are isolated as potential diploids. Three properties of these isolates establish their diploid nature: (1) the isolates are wild type for nutrition and morphology; (2) their conidial length is uniformly greater than that of the haploids (1.21 times); (3) the isolates produce segregants with nonparental combinations of the marker genes. The diploid isolates are much more stable than heterokaryons. The recombinants from the diploids are still diploid, since (1) their conidial length falls in the diploid range, and (2) one of the recombinants has segregated a second-order recombinant. Many of the expected classes of recombinants have not been detected.     

Genetic linkage map for Amylostereum areolatum reveals an association between vegetative growth and sexual and self-recognition

Amylostereum areolatum is a filamentous fungus that grows through tip extension, branching and hyphal fusion. In the homokaryotic phase, the hyphae of different individuals are capable of fusing followed by heterokaryon formation, only if they have dissimilar allelic specificities at their mating-type (mat) loci. In turn, hyphal fusion between heterokaryons persists only when they share the same alleles at all of their heterokaryon incompatibility (het) loci. In this study we present the first genetic linkage map for A. areolatum, onto which the mat and het loci, as well as quantitative trait loci (QTLs) for mycelial growth rate are mapped. The recognition loci (mat-A and het-A) are positioned near QTLs associated with mycelial growth, suggesting that the genetic determinants influencing recognition and growth rate in A. areolatum are closely associated. This was confirmed when isolates associated with specific mat and het loci displayed significantly different mycelial growth rates. Although the link between growth and sexual recognition has previously been observed in other fungi, this is the first time that an association between growth and self-recognition has been shown.     

Indole- and caffeine-resistant mutations of Schizophyllum commune are involved in the behavior of a class III B mating-type factor in trp1 cells

We have reported that a parental strain of Schizophyllun commune T11 (trp1) is fully compatible with a strain T37 (Bα1′β4, trp1), but indole- and caffeine-resistant mutants (In^R-13 and Caf^R-9 respectively) derived from the T11 are semicompatible with the same T37. The compound-resistant mutations, ind1 and cfn1, were linked to neither A nor B mating-type factors. In^R-13, Caf^R-9, and all of indole- and caffeine-resistant progenies (ind1, trp1; cfn1, trp1) were semicompatible with the T37, and compatible with a strain T40 (Bα1′β4, TRP1) although the T40 contained the same class III B mating-type factor as the T37. The In^R-13 and Caf^R-9 were also semicompatible with any tester strains (Bα1′β4, trp1) developed, and the resulting heterokaryons produced non-aggregated and semiaggregated masses of hyphae, respectively. In the mutant heterokaryons, nuclei were distributed irregularly especially in case of [trp1/trp1] background; anucleate, uninucleate, binucleate and multinucleate cells were found with modified hyphae which contained a different type of septation (pseudoclamps and simple septa other than true clamps). We concluded that the ind1 and cfn1 mutations alter the normal behavior of one of class III B mating-type factors in Trp⁻ cells.     

Suppressive effect of protease inhibitors on heterokaryons containing chick erythrocyte nuclei

Nuclei of active cells (HeLa, mouse fibroblasts) partnered with chick erythrocyte nuclei in heterokaryons are suppressed, as judged by a decreased rate of ³H-uridine incorporation and diminished nuclear binding of ³H-actinomycin D. The extent to which active partner nuclei are suppressed, the extent to which erythrocyte nuclei are reactivated, and the degree of sensitivity of heterokaryons towards certain inhibitors of proteolytic enzymes, all correlate strongly with the ratios of erythrocyte nuclei to active nuclei. Thus, reactivation of individual erythrocyte nuclei is reduced progressively and active nuclei are suppressed progressively as the ratio of erythrocyte nuclei per active nucleus in heterokaryons increases. This erythrocyte nuclear-dose dependent suppression is markedly amplified when heterokaryons are grown in the presence of protease inhibitors. The protease inhibitors found to affect heterokaryons are low molecular weight (<400) inhibitors of trypsin-like enzymes: -1-tosylamide-2-leucyl chloromethyl ketone (TLCK), N-α-tosyl- -arginine methyl ester (TAME) and N-benzoyl- -arginine amide (BAA). They affect heterokaryons at concentrations comparable to the minimal concentrations at which they inhibit trypsin. Nonfused HeLa cells, mouse fibroblasts, or their homokaryons are refractory to protease inhibitors at these concentrations.Reactivation of chick erythrocyte nuclei in a heterokaryon may involve release of suppressors ordinarily confined to the erythrocyte nucleus, with subsequent redistribution of suppressor among all the nuclei of the heterokaryon. Under these circumstances the state of nuclear activity will depend on the quantity of suppressor per individual nucleus; within the erythrocyte nucleus the suppressors will decrease its rate of reactivation, when they migrate into an active nucleus they will suppress it. These suppressors, either in transit between the nuclei, or within the nuclei, may be hydrolysed by intracellular proteases.     

Slow-growing heterokaryons as potential intermediates in supernumerary chromosome transfer between biotypes of Colletotrichum gloeosporioides

The vegetative transfer of a supernumerary chromosome between two biotypes (A and B) of Colletotrichum gloeosporioides pathogenic on Stylosanthes spp. has been previously demonstrated. The mechanism of transfer is unknown, and transient heterokaryosis has been implicated as an intermediary step, but inter-biotype heterokaryons have not so far been isolated. Conidia of a hygromycin-resistant strain of biotype A and a phleomycin-resistant strain of biotype B were mixed, co-cultured and plated on media containing both antibiotics and extremely slow-growing colonies resistant to both antibiotics isolated using repeated hyphal tip sub-culturing. Mononucleate conidia derived from these colonies were unable to germinate on media containing both antibiotics, but were able to germinate on media containing only one antibiotic, with hygromycin-resistant colonies predominating, indicating that unbalanced heterokaryons had formed. The heterokaryons had highly impaired growth rates suggesting some incompatibility. DNA marker analysis confirmed their heterokaryon status. These results demonstrate that unfit inter-biotype heterokaryons can form and potentially provide an intermediate step for supernumerary chromosomal exchange.     

Heterokaryosis inFusarium moniliforme

Ultraviolet light readily induced both auxotrophic and morphological mutants in isolates ofF. moniliforme from corn and sorghum. Sexual analysis of these mutants yielded a genetic map with several instances of linkage. Forced heterokaryons formed readily between auxotrophs of the same strain but not between auxotrophs of different strains. At least four nuclear gene loci, but not the mating-type locus, controlled heterokaryon formation in this fungus. In the auxotroph pairings, heterokaryosis was confined to the anastomosed cells; there was no evidence for nuclear migration through established hyphae. Genetic complementation occurred in anastomosed cells, and the products of this complementation were then translocated to the homokaryotic cells of the colony. No heterozygous diploids were recovered from the heterokaryons, although there was evidence for rare somatic gene recombination. Heterokaryosis in isolates ofF. moniliforme in nature is probably rare.     

Rare-Cell Fusion Events between Diploid and Haploid Strains of the Sexually Agglutinative Yeast HANSENULA WINGEI

Diploids of the yeast Hansenula wingei are nonagglutinative and do not form zygotes in mixed cultures with either sexually agglutinative haploid mating type. However, a low frequency of diploid x haploid cell fusions (about 10^-3) is detectable by prototrophic selection. This frequency of rare diploid x haploid matings is not increased after the diploid culture is induced for sexual agglutination. Therefore, we conclude that genes that repress mating are different from those that repress sexual agglutination.——Six prototrophs isolated from one diploid x haploid cross had an average DNA value (µg DNA per 10⁸ cells) of 6.19, compared to 2.53 and 4.35 for the haploid and diploid strains, respectively. Four prototrophs were clearly cell-fusion products because they contained genes from both the diploid and the haploid partners. However, genetic analysis of the prototrophs yielded results inconsistent with triploid meiosis; all six isolates yielded a 2:2 segregation for the mating-type alleles and linked genes.——Mitotic segregation of monosomic (2n-1) cells lacking one homolog of the chromosome carrying the mating-type locus is proposed to explain the rare production of sexually active cells in the diploid cultures. Fusion between such monosomic cells and normal haploids is thought to have produced 3n-1 cells, disomic for the chromosome carrying the mating-type locus. We conclude that in the diploid strain we studied, the physiological mechanisms repressing sexual agglutination and conjugation function efficiently, but events occuring during mitosis lead to a low frequency of genetically altered cells in the population.