Heterozygous diploids ofPenicillium Chrysogenum and their segregation patterns

Several heterozygous diploids were made between genetically labelled derivatives of two strains ofPenicillium chrysogenum which produced relatively large amounts of penicillin and were of divergent lineage. The derivatives were labelled with spore colour and nutritional mutations. The diploids, although uniform in having wild type spore colour and being prototrophic, ranged from types having penicillin yields close to that of the original parents to types having less than a quarter of this titre level. Intermediate types had titre levels of about half to threequarters that of the high yielding diploids. Segregants were selected which had arisen naturally and also after nitrogen mustard treatment; most had the spore colour and auxotrophic phenotype of one or other immediate parent. From diploids of low and intermediate titre only haploid segregants with the genetical markers of one parent could be recovered with intact penicillin yield; haploids with the genetic markers of the other showed a marked reduction in yield. However, from diploids of high yield, both parental types could be recovered showing no loss of their original penicillin yield. The bearing of these results is discussed on the suggestion that different degrees of homozygosity between diploids may account for the titre variation observed. An alternative suggestion that mutations suppressive to penicillin titre might cause such variation is also considered.     

Improvement of Enzyme Productivities through Mutation or Haploidization of Heterozygous Diploids Obtained by Protoplast-fusion of Aspergillus sojae

A new breeding process for Aspergillus sojae involving protoplast fusion was studied to obtain more desirable koji-molds for soy-sauce production, especially as to their enzyme productivities. A pair of double-marker mutants with characteristic enzyme-productivities, derived from genealogically unrelated A. sojae cultures, were fused by means of the protoplast fusion technique. The fusants were UV-treated, and stable heterozygous diploids were obtained. Further improvement of their enzyme-productivities was attempted using two approaches. Through the usual mutation of the heterozygous diploids, the activities of protease and glutaminase were simultaneously improved to a certain extent. Through the haploidization of the heterozygous diploids with benomyl or p-fluorophenylalanine (FPA), some haploids considered phenotypically to be recombinants were segregated, in addition to a large number of haploids that had reverted to the original double marker mutants. On screening these haploid-recombinants, the authors obtained a few more excellent recombinants, as to their enzyme productivities, that is, strains producing as much protease as the hyper-protease producer and producing almost as much glutaminase as the hyper- glutaminase producing parent. It has been shown that mutation or especially haploidization of heterozygous diploids produced by cell-fusion could be a very useful technique for the breeding of new industrial koji-molds.     

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.     

A note on crossing experiments with Aspergillus niger for the production of calcium gluconate

A strong calcium gluconate-producing strain of Aspergillus niger (MN181) was obtained by way of mutagenic treatment. Its growth was very slow with moderate sporulation. The strain was treated with N-methyl-N'nitro-N-nitrosoguanidine (MNNG) and some auxotrophic mutants were obtained. All were less productive than the parent strain in producing calcium gluconate. The reduced yield was corrected in the heterokaryons and diploids derived by crossing sister strains. One diploid strain (D4), heterozygous for auxotrophy and conidial colour markers was grown in the presence of 4% alcohol and 31 segregants were isolated which included both haploid and diploid strains. Their yields were studied and some recombinants were obtained which, in spite of the same yield of MN181, showed improvement in giving fast growth and abundant sporulation.     

Properties of heterozygous diploids between strains ofPenicillium chrysogenum selected for high penicillin yield

Three strains ofPenicillium chrysogenum selected for high penicillin yield and of independent lineage were marked with suitable genetical characters prior to the synthesis of several heterozygous diploids. These parental strains had domestic codes, C, D and Y. Two diploids, between differently labelled mutants of strain C and Y, produced similar amounts of penicillin to strain C, which was less than that produced by strain Y. Previous work had indicated that genes responsible for increased penicillin yield were recessive and the present results suggested that such genes in strains C and Y were allelic, apart from the presence of one or more additional recessive mutations leading to greater penicillin production in the higher yielding parent. Three diploids made between mutants of strains D and Y were lower in penicillin yield than either original parent and only in the case of one diploid compared with one of the parental strains was this difference not significant. In strains D and Y, therefore, there may have been some recessive genes concerned with increasing penicillin yield which were non-allelic. However, no first order segregants arising spontaneously or subsequent to X-ray treatment produced higher levels of penicillin than the better yielding original parent in any cross.     

Chromosomal behavior of anther culture derived plants of rice

Cytological examination of anther-culture-derived plants of rice showed that the regenerants were predominantly diploids and haploids. Haploid meiosis indicated that the earlier hypothesis of rice being an ancient polyploid is unlikely. Diploids generally were normal and fertile. The low frequency of polyploids (1.5%) probably was due to rapid regeneration of plants from short term callus cultures.     

Development of porcine blastocysts from in vitro- matured and activated haploid and diploid oocytes

The purpose of this study was to determine the developmental capacity of electro-activated porcine oocytes. Follicular oocytes collected from gilts at local slaughterhouses were matured for 48 h and were then subjected to a single square pulse of direct current for 100 rhojusec at 1,500 V/cm for activation. To obtain activated diploid oocytes, some were treated with 5.0 micro/ml cytochalasin B for 4 h immediately after electro-activation. The frequency of activation ranged from 96 to 100%. While 91% of activated oocytes that had not been treated with cytochalasin B had 2 polar bodies and a nucleus (haploids), 92% of the oocytes treated with cytochalasin B had only the first polar body and 2 nuclei (diploids). Haploids and diploids were further cultured in TCM-199 medium that contained 10% (v/v) heat- treated fetal calf serum (FCS) and 0.1 mg/ml sodium pyruvate (mTCM) or in Whittenk medium plus 0.4% (w/v) bovine serum albumin (BSA). The frequency of abnormal oocytes was significantly higher in mTCM (83%) than in Whitten's medium (65%) 96 h after the electro-activation (P < 0.01), suggesting that Whitten's medium supported the development of activated oocytes beyond the morula stage. In all cases, several oocytes developed to the blastocyst stage 144 h after electro- activation (1 to 12%). The frequency was significantly higher in the case of diploids cultured in Whitten's medium (12%) (P < 0.01) than in the case of haploids cultured in Whitten's medium (4%), or in the case of haploids cultured in mTCM (1%). The mean number of nuclei per blastocyst was significantly lower in mTCM (haploids, 15; diploids, 16.1) than in Whitten's medium (haploids, 35.7; diploids, 40.1; P < 0.01), suggesting that the number of nuclei in blastocysts was affected by the culture medium.     

Visualizing nuclear migration during conidiophore development in Aspergillus nidulans and Aspergillus oryzae: multinucleation of conidia occurs through direct migration of plural nuclei from phialides and confers greater viability and early germination in Aspergillus oryzae

Nuclear migration is indispensable for normal growth, differentiation, and development, and has been studied in several fungi including Aspergillus nidulans and Neurospora crassa. To better characterize nuclear movement and its consequences during conidiophore development, conidiation, and conidial germination, we performed confocal microscopy and time-lapse imaging on A. nidulans and Aspergillus oryzae strains expressing the histone H2B-EGFP fusion protein. Active trafficking of nuclei from a vesicle to a phialide and subsequently into a conidium provided the mechanistic basis for the formation of multinucleate conidia in A. oryzae. In particular, the first direct visual evidence on multinucleate conidium formation by the migration of nuclei from a phialide into the conidium, rather than by mitotic division in a newly formed conidium, was obtained. Interestingly, a statistical analysis on conidial germination revealed that conidia with more nuclei germinated earlier than those with fewer nuclei. Moreover, multinucleation of conidia conferred greater viability and resistance to UV-irradiation and freeze-thaw treatment.     

A note on crossing experiments with Aspergillus niger for the production of calcium gluconate

K undu , P.N. & D as , A. 1985. A note on crossing experiments with Aspergillus niger for the production of calcium gluconate. Journal of Applied Bacteriology 59 , 1–5.
A strong calcium gluconate-producing strain of Aspergillus niger (MN181) was obtained by way of mutagenic treatment. Its growth was very slow with moderate sporulation. The strain was treated with N-methyl-N-nitro-N-nitrosoguanidne (MNNG) and some auxotrophic mutants were obtained. All were less productive than the parent strain in producing calcium gluconate. The reduced yield was corrected in the heterokaryons and diploids derived by crossing sister strains. One diploid strain (D4), heterozygous for auxotrophy and conidial colour markers was grown in the presence of 4% alcohol and 31 segregants were isolated which included both haploid and diploid strains. Their yields were studied and some recombinants were obtained which, in spite of the same yield of MN181, showed improvement in giving fast growth and abundant sporulation.     

A mutant induced in the malting barley cv Triumph with reduced dormancy and ABA response

 Induced mutants in the barley cultivar Triumph have been screened for reduced dormancy. One line, which germinated readily 2 weeks after harvest, was classified as ABA-insensitive, since it could tolerate a ten-fold increase in ABA, compared to its parent, before germination was inhibited. This mutant, designated TL43, was genotypically similar to Triumph and phenotypically similar under Scottish growing conditions, except for a slightly reduced grain size. In Spain, it showed considerable reductions in both grain yield and plant height, suggesting that it was less widely adapted than its parent. Levels of α-amylase activity were increased at both sites. The mutant appeared to be different from those with ABA insensitivity or altered dormancy previously documented in either barley or Arabidopsis. Received: 23 March 1998 / Accepted: 13 August 1998     

Successful induction and recognition of conidiation,conidial germination and chlamydospore formation in pure culture of Morchella

Morels, fungi from the genus Morchella, are popular edible mushrooms. However, little knowledge of their asexual reproduction and inaccessible pure mitospores hamper illumination of their life cycle. Herein, we successfully induced conidiation, conidial germination and chlamydospore formation in pure culture of Morchella sextelata. Conidiation proceeded via four morphologically distinct stages: development of the conidiophore stalk, stalk branching, phialide differentiation, and conidium production. Terminal and intercalary chlamydospores were formed on conidial hyphae. The development of conidiophores occurred earlier, with more conidia produced, in cross-mating cultures than in single-spore cultures. Mature conidia were spherical and 2.5–8 μm in diameter, with a vast majority (nearly 99%) 2.5–5 μm in diameter. Each conidium contained one to three nuclei (80.2% conidia contained one nucleus, 19.1% contained two nuclei, and 0.7% contained three nuclei). The conidial nucleus diameter was 1–2 μm. The nuclear mitosis in detached conidia that was observed may benefit their colony initiation. Additionally, morel conidia formed conidial anastomosis tubes. Conidia (mitospores) likely not only function as spermatia, but also as reproductive propagules in Morchella. Further research is imperative to elucidate the relationship between the conidia and chlamydospores, and their unique function in the morel life cycle.     

Dominant spore color mutants of Aspergillus nidulans defective in germination and sexual development.

The ascomycete Aspergillus nidulans produces green conidia (asexual spores). Recessive mutants which produce yellow conidia have been previously isolated from haploid strains and have been shown to be deficient in laccase (diphenol oxidase), an enzyme that requires copper for activity. Using a diploid parent strain, we isolated dominant yellow conidial mutants which, in the haploid state, produced even less laccase activity than a recessive mutant. Three isolates of such mutants behaved similarly and define a single complementation group (yB) on chromosome VIII distinct from the yA locus on chromosome I defined by recessive mutants. Unlike yA mutants, whose only discernable phenotype is their conidial color, yB mutants are pleiotropic: conidial germination was delayed relative to the wild type, and sexual development was blocked at an early stage. The three phenotypes of yB mutants were expressed on yeast extract-glucose medium containing 1.6 microM of added copper. When copper was added to above 5 microM, all three phenotypes were remediated, and near wild-type levels of laccase were produced. We conclude that yB mutants have a reduced availability of copper. The dominance of yB mutants could result, for example, from an alteration in transport or storage of copper. Using an immunological assay, we detected no laccase antigenic cross-reacting material in yB mutants grown on medium of low copper content. We conclude that either the synthesis or the stability of laccase is copper dependent.     

Haplo-insufficiency for different genes differentially reduces pathogenicity and virulence in a fungal phytopathogen

The main determinant of pathogenicity in Ustilago maydis is the b-mating locus, where establishment of heterozygosity is sufficient to cause galls/tumors on maize plants. However, matings between haploids where one partner contains a mutation, in e.g., the smu1 gene, encoding a Ste20-like PAK kinase, often show reduced mating and pathogenicity compared to wild type. Here we show that similarly, diploids lacking one copy of smu1, are reduced in production of aerial hyphae, but do not show significantly-reduced virulence. Haplo-insufficiency was also observed for additional genes. UmPde1 is a cyclic phosphodiesterase involved in cAMP turnover as part of the cAMP-dependent PKA pathway. Hsl7 plays a role in cell length and in the filamentous response to low ammonium in haploid cells. Diploids deleted for one copy of either the pde1 or hsl7 genes had reduced or increased production of aerial hyphae, respectively, and both were severely impaired in virulence compared to wild type diploids. rho1 and pdc1 are two genes essential for cell viability in haploids. These genes also displayed haplo-insufficiency for pathogenesis. rho1/Δrho1 diploid cells were defective in pheromone production and detection, aerial hyphae induction, and were avirulent. In contrast, pdc1/Δpdc1 diploid cells only failed to produce tumors when applied to maize whorls. We predict the haplo-insufficiency of most of these signaling components is due to stoichiometric imbalance of the respective gene products with their interacting partners, thereby impairing virulence-induction mechanism(s). Further investigation of the bases for such haplo-insufficiency as well as of additional genes displaying this phenotype will provide important insights into fundamental aspects of development in this organism as well as inter-nuclear communication and genetic control.     

酿酒酵母呼吸缺陷型和野生型酒精发酵特性的比较分析*

比较了酒精发酵生产菌株IFFI1300及其呼吸缺陷型突变株在酒精产量、发酵动力学、耐酒精能力及与酒精发酵相关的乙醇脱氢酶活性等方面的特性。结果表明：1)发酵终期的酒精产量,45株呼吸缺陷型的平均值与野生型没有显著性差异;但部分缺陷型的酒精产量高于野生型。2)酒精发酵动力学结果显示,呼吸缺陷型酒精产生速度略高于野生型。3)单位重量干菌体的乙醇脱氢酶活性,呼吸缺陷型高于野生型。以上结果提示：呼吸缺陷型用于酒精发酵以提高酒精产量和缩短发酵周期是有潜力的。4)单位体积发酵液的乙醇脱氢酶活性则野生型高于呼吸缺陷型,主要原因在于呼吸缺陷型的生物量明显低于野生型。5)呼吸缺陷型菌株之间的耐酒精能力差别很小,耐酒精能力的高低与酒精产量的高低没有明显的正相关性。一般的,酒精产量高的菌株耐酒精能力较强。在实验结果的基础上,对呼吸缺陷型用于酒精发酵的优越性和可行性进行了讨论。     

Genetic analysis of polyauxotrophy and early mitotic progeny of Saccharomyces cerevisiae zygotes. II. Spore-forming segregants in the mitotic and meiotic progeny of polyauxotrophic clones

This article continues the investigation of polyauxotrophic (PA) clones formed in early mitotic progeny of zygotes. Cloning and segregation analysis of PA progeny suggest an unusual state of diploid genome in these strains, which is expressed as elimination of the dominance effect of the wild allele and as suppression or conversion of either of two loci of mating type. In PA progeny, except for recombinant haploids, sporulating diploids and unstable clones were detected. The tetrad analysis of the diploids points to homozygotization for individual markers. Over-replication of diploid set of chromosomes, prior to meiosis, and replacement of the haploid nucleus (the product of meiosis) for the diploid nucleus may explain the appearance of sporulating segregants in the diploid meiotic progeny. Unstable segregants may be considered as heterokaryons with complex interaction of nuclei.     

Gluconic acid production by Aspergillus niger mutant ORS-4.410 in submerged and solid state surface fermentation

Aspergillus niger ORS-4.410, a mutant of Aspergillus niger ORS-4 was produced by repeated irradiation with UV rays. Treatments with chemical mutagnes also resulted into mutant strains. The mutants differed from the parent strain morphologically and in gluconic acid production. The relationship between UV treatment dosage, conidial survival and frequency of mutation showed the maximum frequency of positive mutants (25%) was obtained along with a conidial survival of 59% after second stage of UV irradiation. Comparison of gluconic acid production of the parent and mutant ORS-4.410 strain showed a significant increase in gluconic acid production that was 87% higher than the wild type strain. ORS-4.410 strain when transferred every 15 days and monitored for gluconic acid levels for a total period of ten months appeared stable. Mutant ORS-4.410 at 12% substrate concentration resulted into significantly higher i.e. 85-87 and 94-97% yields of gluconic acid under submerged and solid state surface conditions respectively. Further increase in substrate concentration appeared inhibitory. Maximum yield of gluconic acid was obtained after 6 days under submerged condition and decreased on further cultivation. Solid state surface culture condition on the other hand resulted into higher yield after 12 days of cultivation and similar levels of yields continued thereafter.     

Genetics of Aspergillus flavus: linkage of aflatoxin mutants

Eight aflatoxin (afl) mutants of Aspergillus flavus were induced with N-methyl-N'-nitro-N-nitrosoguanidine. Heterozygous diploids formed between afl mutants and tester strains revealed that each afl mutant was recessive. Haploids selected from these heterozygous diploids indicated the linkage of all eight afl mutants to markers on group VII. These include previously mapped arg-7 (arginine), leu (leucine), dominant afl-1, and nor which accumulates norsolorinic acid that is visible as an orange-red pigment. Diploid complementation tests indicated that all but two afl mutants were nonallelic. Diploids homozygous for nor, resulting from crossing-over, were isolated and used to map new afl genes.     

Construction of novel tumor necrosis factor-alpha mutants with reduced toxicity and higher cytotoxicity on human tumor cells

Two tumor necrosis factor-α mutants MT1 (32Trp157Phe) and MT2 (2Lys30Ser-32Trp 157Phe) were constructed by site-directed mutagenesis. These mutants were soluble and over-expressed inE. coli. The purity of purified mutants was above 95% by serial chromatography. The results of Western blot indicated that these mutants could be cross-reactive with monoclonal antibody against native hTNF-α. Compared to parent hTNF-α, the cytotoxicity of these mutants on murine fibrosarcoma L929 cell lines reduced 4–5 orders of magnitude but was equivalent to that of native hTNF-α on human tumor cell lines. The LD50 of mutant MT1 was reduced to 0.34% of wild type and the dose of MT2 that resulted in 30% death of mice reduced to less than 1/700 that of parent hTNF-α.     

单套染色体组在泽蛙雌核单倍体发育中的作用

本文比较了泽蛙(Rana limnocharis Boie)单倍体和二倍体的胚胎发育。结果表明:泽蛙单倍体的生活力低下,器官发生异常,自原肠胚起发育速度减慢。据此,作者讨论了单套染色体组在个体发育中的作用。     

Multinucleation in the conidia of polyploids derived fromTrichoderma reesei QM 9414 by colchicine treatment

Conidia ofTrichoderma reesei QM 9414 were treated with colchicine in order to obtain polyploids (diploids; tetraploids). Cellulase production by diploids (mononucleate conidia) was almost twice as great as that of the original strain, but that of tetraploids (binucleate conidia) was not increased. When these latter conidia were re-treated with 2.0% (w/v) colchicine, multiple nuclei were produced in each conidium, and their diameter was almost the same as that of the original nucleus. Cellulase production of the diploid was almost the same in either mononucleate or multinucleate nature. However, cellulase production by the tetraploid which produced multinucleate conidia was greater than that of the binucleate tetraploid and that of the diploid. The multinucleation technique can contribute to enhancing cellulase production.