Os-1 Mutant Research Articles

Adaptative responses of Neurospora crassa by histidine kinases upon the attack of the arthropod Sinella curviseta.

Histidine kinases (HKs) are important sensor proteins in fungi and play an essential role in environmental adaptation. However, the mechanisms by which fungi sense and respond to fungivores attack via HKs are not fully understood. In this study, we utilized Neurospora crassa to investigate the involvement of HKs in responding to fungivores attack. We found that the 11 HKs in N. crassa not only affected the growth and development, but also led to fluctuations in antioxidant production. Ten mutants in the genes encoding HKs (except ∆phy1) showed increased production of reactive oxygen species (ROS), especially upon Sinella curviseta attack. The ROS burst triggered changes in conidia and perithecial beaks formation, as well as accumulation of β-glucan, ergothioneine, ergosterol, and carotenoids. β-glucan was increased in ∆hk9, ∆os1, ∆hcp1, ∆nik2, ∆sln1, ∆phy1 and ∆phy2 mutants compared to the wild-type strain. In parallel, ergothioneine accumulation was improved in ∆phy1 and ∆hk16 mutants and further increased upon attack, except in ∆os1 and ∆hk16 mutants. Additionally, fungivores attack stimulated ergosterol and dehydroergosterol production in ∆hk9 and ∆os1 mutants. Furthermore, deletion of these genes altered carotenoid accumulation, with wild-type strain, ∆hk9, ∆os1, ∆hcp1, ∆sln1, ∆phy2, and ∆dcc1mutants showing an increase in carotenoids upon attack. Taken together, HKs are involved in regulating the production of conidia and antioxidants. Thus, HKs may act as sensors of fungivores attack and effectively improve the adaptive capacity of fungi to environmental stimuli.

OS1 functions in the allocation of nutrients between the endosperm and embryo in maize seeds.

Uncovering the genetic basis of seed development will provide useful tools for improving both crop yield and nutritional value. However, the genetic regulatory networks of maize (Zea mays) seed development remain largely unknown. The maize opaque endosperm and small germ 1 (os1) mutant has opaque endosperm and a small embryo. Here, we cloned OS1 and show that it encodes a putative transcription factor containing an RWP-RK domain. Transcriptional analysis indicated that OS1 expression is elevated in early endosperm development, especially in the basal endosperm transfer layer (BETL), conducting zone (CZ), and central starch endosperm (CSE) cells. RNA sequencing (RNA-Seq) analysis of the os1 mutant revealed sharp downregulation of certain genes in specific cell types, including ZmMRP-1 and Meg1 in BETL cells and a majority of zein- and starch-related genes in CSE cells. Using a haploid induction system, we show that wild-type endosperm could rescue the smaller size of os1 embryo, which suggests that nutrients are allocated by the wild-type endosperm. Therefore, our data imply that the network regulated by OS1 accomplishes a key step in nutrient allocation between endosperm and embryo within maize seeds. Identification of this network will help uncover the mechanisms regulating the nutritional balance between endosperm and embryo.

Mutations in an amino acid transporter gene are responsible for sex-linked translucent larval skin of the silkworm, Bombyx mori

The sex-linked translucent ( os) mutation in the silkworm, Bombyx mori, confers slightly translucent larval skin resulting from a decrease in the incorporation of uric acid into epidermal cells. By positional cloning, we narrowed a region linked to the os phenotype to approximately 157 kb located on scaffold Bm_scaf72 on the Z chromosome (chromosome 1). The region contained four gene models. Sequencing analysis revealed that one of the candidate genes had a 7-bp deletion in the coding region. We also found a 111-bp deletion or single-nucleotide substitution in the same gene using independent os mutant strains. Because all the mutations caused the generation of abnormal transcripts followed by translation of a truncated protein, we conclude that the mutation of this candidate gene is responsible for the translucent larval skin of the os mutant. Sequence analysis indicated that the gene responsible for the os mutation had homology to amino acid transporters of the solute carrier family of proteins. Our results suggest that solute carrier proteins are involved in uric acid transport in insects and other invertebrates.

Maturational Alteration of Oppositely Orientated rDNA and Differential Proliferation of GC- and AT-biased Genotypes of Ophiocordyceps sinensis and Paecilomyces hepiali in Natural Cordyceps sinensis

Multiple reports have detailed the simultaneous detection of ribosomal DNA (rDNA) of Paecilomyces hepiali (Ph), Hirsutella sinensis and two genotypes of Ophiocordyceps sinensis (Os) from Cordyceps sinensis (Cs), and the altered phenotypes and chemical components of Cs during Cs maturation. In this study, we observed an increase in the biomass of Ph and two moieties of Os rDNA during Cs maturation. The GCand AT-biased genotypes of transition mutations in the Cs stroma were confirmed using MassARRAY SNP genotyping. In premature Cs, the AT-biased genotypes were not expressed in the caterpillar, but highly predominated in the stroma. The GC bias was expressed in an opposite manner in the premature Cs compartments. The differential expression of GC- and AT-biased mutants was altered during Cs maturation. The increased biomass of the inverse-oriented Os rDNA of the GC-genotype was associated with a maturational increase in stroma height. We also report evidence of transversion mutations within Cs genes. The dynamic alterations of Ph and Os mutant gene expressions along with Cs maturation may play an essential role in Cs germination and maturation, the key elements of Cs life cycle, and result in the varied therapeutic potency of Cs.

The Response Regulator RRG-1 Functions Upstream of a Mitogen-activated Protein Kinase Pathway Impacting Asexual Development, Female Fertility, Osmotic Stress, and Fungicide Resistance inNeurospora crassa

Two-component systems, consisting of proteins with histidine kinase and/or response regulator domains, regulate environmental responses in bacteria, Archaea, fungi, slime molds, and plants. Here, we characterize RRG-1, a response regulator protein from the filamentous fungus Neurospora crassa. The cell lysis phenotype of Delta rrg-1 mutants is reminiscent of osmotic-sensitive (os) mutants, including nik-1/os-1 (a histidine kinase) and strains defective in components of a mitogen-activated protein kinase (MAPK) pathway: os-4 (MAPK kinase kinase), os-5 (MAPK kinase), and os-2 (MAPK). Similar to os mutants, Delta rrg-1 strains are sensitive to hyperosmotic conditions, and they are resistant to the fungicides fludioxonil and iprodione. Like os-5, os-4, and os-2 mutants, but in contrast to nik-1/os-1 strains, Delta rrg-1 mutants do not produce female reproductive structures (protoperithecia) when nitrogen starved. OS-2-phosphate levels are elevated in wild-type cells exposed to NaCl or fludioxonil, but they are nearly undetectable in Delta rrg-1 strains. OS-2-phosphate levels are also low in Delta rrg-1, os-2, and os-4 mutants under nitrogen starvation. Analysis of the rrg-1(D921N) allele, mutated in the predicted phosphorylation site, provides support for phosphorylation-dependent and -independent functions for RRG-1. The data indicate that RRG-1 controls vegetative cell integrity, hyperosmotic sensitivity, fungicide resistance, and protoperithecial development through regulation of the OS-4/OS-5/OS-2 MAPK pathway.

Roles of putative His-to-Asp signaling modules HPT-1 and RRG-2, on viability and sensitivity to osmotic and oxidative stresses in Neurospora crassa

Neurospora crassa has a putative histidine phosphotransfer protein (HPT-1) that transfers signals from 11 histidine kinases to two putative response regulators (RRG-1 and RRG-2) in its histidine-to-aspartate phosphorelay system. The hpt-1 gene was successfully disrupted in the os-2 (MAP kinase gene) mutant, but not in the wild-type strain in this study. Crossing the resultant hpt-1; os-2 mutants with the wild-type or os-1 (histidine kinase gene) mutant strains produced no progeny with hpt-1 or os-1; hpt-1 mutation, strongly suggesting that hpt-1 is essential for growth unless downstream OS-2 is inactivated. hpt-1 mutation partially recovered the osmotic sensitivity of os-2 mutants, implying the involvement of yeast Skn7-like RRG-2 in osmoregulation. However, the rrg-2 disruption did not change the osmotic sensitivity of the wild-type strain and the os-2 mutant, suggesting that rrg-2 did not participate in the osmoregulation. Both rrg-2 and os-2 single mutation slightly increased sensitivity to t-butyl hydroperoxide, and rrg-2 and hpt-1 mutations increased the os-2 mutant's sensitivity. Although OS-1 is considered as a positive regulator of OS-2 MAP kinase, our results suggested that HPT-1 negatively regulated downstream MAP kinase cascade, and that OS-2 and RRG-2 probably participate independently in the oxidative stress response in N. crassa.

Involvement of OS-2 MAP kinase in regulation of the large-subunit catalases CAT-1 and CAT-3 in Neurospora crassa

Neurospora crassa has four catalase genes--cat-1, cat-2, cat-3, and ctt-1/cat-4. cat-1 and cat-3 encode two fungal-specific large-subunit catalases CAT-1 and CAT-3 normally produced in conidia and growing hyphae, respectively. cat-2 encodes CAT-2 catalase-peroxidase normally produced in conidia. ctt-1 (or cat-4), of which expression was controlled by OS-2 MAP kinase (Noguchi et al., Fungal Genet. Biol. 44, 208-218), encodes a small-subunit catalase with unknown function. To clarify the contribution of OS-2 on the regulation of CAT-1, CAT-2, and CAT-3, we performed quantitative RT-PCR and in-gel catalase activity analyses. When the hyphae were treated with a fungicide (1 mug/ml fludioxonil) or subjected to an osmotic stress (1 M sorbitol), cat-1 was strongly upregulated and CAT-1 was reasonably induced in the wild-type strain. Interestingly, fludioxonil caused not only the CAT-1 induction but also a remarkable CAT-3 decrease in the wild-type hyphae, implying of an abnormal stimulation of asexual differentiation. These responses were not observed in an os-2 mutant hyphae, indicating an involvement of OS-2 in the cat-1 expression; however, os-2 was dispensable for the production of CAT-1 in conidia. In contrast, the expression of cat-2 was significantly induced by heat shock (45 degrees C) and that of cat-3 was moderately stimulated by an oxidative stress (50 microg/ml methyl viologen) in both the wild-type strain and the os-2 mutant, and corresponding enzyme activities were detected after the treatments. Although basal levels of transcription of cat-1 and cat-3 in an os-2 mutant hyphae were a few-fold lower than in the wild-type hyphae, the os-2 mutant exhibited a considerably lower levels of CAT-3 activity than the wild-type strain. These findings suggest that OS-2 MAP kinase regulated the expression of cat-1 and cat-3 transcriptionally, and probably that of cat-3 posttranscriptionally, even though the presence of another regulatory system for each of these two genes is evident.

Identification of OS-2 MAP kinase-dependent genes induced in response to osmotic stress, antifungal agent fludioxonil, and heat shock in Neurospora crassa

Two-component signal transduction comprising of OS-1 (histidine kinase), OS-4 (MAPKK kinase), OS-5 (MAPK kinase), and OS-2 (MAP kinase) plays an important role in osmotic regulation in Neurospora crassa. To identify the genes regulated downstream of OS-2 MAP kinase, quantitative real-time RT-PCR analysis was conducted in selected genes based on Hog1 MAP kinase regulated genes in yeast. In response to osmotic stress and fludioxonil, expression of six genes that for glycerol synthesis ( gcy-1, gcy-3, and dak-1), gluconeogenesis ( fbp-1 and pck-1), and catalase ( ctt-1) was activated in the wild-type strain, but not in the os-2 mutant. A heat shock treatment also induced their expression in the same way. Consisting with the gene expression, the enzyme activity of glycerol dehydrogenase, but not glycerol-3-phosphate dehydrogenase, was increased in response to osmotic stress and fludioxonil in the wild-type strain. OS-2 was phosphorylated by the OS-1 cascade in response to relatively low osmotic stress and fludioxonil. However, OS-2 phosphorylation by heat shock and a higher osmotic stress was found in the os-1 mutant normally but not in the os-4 and os-5 mutants. These results suggested that non-OS-1 signaling activates OS-2 in an OS-4-dependent manner in such conditions.

Structural and kinetic alterations of constitutive conidial alkaline phosphatase from the osmotically-sensitive mutant ofNeurospora crassa

The osmotically-sensitive os-1 mutant of Neurospora crassa overproduced conidial alkaline phosphatase. The enzyme was purified by Phenyl-Sepharose CL-4B chromatography and Sephadex G-200 gel filtration. PAGE analysis of the purified enzyme suggested the occurrence of aggregation and/or disaggregation phenomena. The enzyme is a glycoprotein containing 16% saccharide, with apparent molar mass of 137 kDa. Two protein bands (36 and 62 kDa) were observed in SDS-PAGE, suggesting that the native enzyme was a trimer. The pI was estimated to be 2.7, and optima of pH and temperature were 9.5 and 65 degrees C, respectively. The enzyme showed broad substrate specificity, hydrolyzing preferentially 4-nitrophenyl phosphate, O-phosphoamino-acids and 2-phosphoglycerate. The hydrolysis of 4-nitrophenyl phosphate was stimulated by Co(II) (26%), Ni(II) (23%) and Mg(II) ions (80%). The enzyme was stable for up to 6 months at 4 degrees C in 5 mmol/L Tris-HCl buffer and also upon storage at 25 degrees C for 10 d. The kinetic and structural properties of the conidial enzyme purified from the os-1 mutant were quite different from those of the wild type strain. The enzyme overproduction observed in the mutant may be related to cell wall alterations that affect the process of enzyme secretion.

Induction of OS-2 phosphorylation in Neurospora crassa by treatment with phenylpyrrole fungicides and osmotic stress

Neurospora crassa is sensitive to phenylpyrrole fungicides and to high osmolarity. By contrast, the osmosensitive mutant os-2 is resistant to this class of fungicides. To understand the relationship between the phenylpyrroles fungitoxicity and the osmoregulatory pathway, we investigated the regulation of the mitogen-activated protein kinase (MAPK) OS-2 in Neurospora. The proteomic tools were used for the characterization of the OS-2 activation and for the measurement of the MAPK cascade induction in situ. Finally, the identification of the genes controlled by os-2 was attempted by comparing the proteomes of the wild type and the os-2 mutant strains. The results indicated that OS-2 is regulated by a dual phosphorylation on a Thr-X-Tyr motif. Furthermore, OS-2 undergoes a rapid phosphorylation after treatment with phenylpyrroles (fenpiclonil and fludioxonil) and under elevated salt concentrations in the surrounding medium. Finally, different enzymes involved in the metabolism of sugars and amino acids were identified as os-2-dependent.

Characterization of mutations in the two-component histidine kinase gene AbNIK1 from Alternaria brassicicola that confer high dicarboximide and phenylpyrrole resistance

Highly iprodione- and fludioxonil-resistant field and laboratory isolates of A. brassicicola were found to be either moderately sensitive or tolerant to osmotic stress. AbNIK1, a two-component histidine kinase gene, was isolated from a fungicide-sensitive strain. The predicted protein possessed the six tandem amino acid repeats at the N-terminal end, which is a landmark of osmosensor histidine kinases from filamentous fungi. A comparison of the nucleic acid sequences of the AbNIK1 gene from fungicide-sensitive and fungicide-resistant isolates revealed the presence of mutations in six of the seven resistant strains analyzed. Null mutants were all found to be moderately sensitive to osmotic stress, indicating that they are similar to Neurospora crassa Type I os-1 mutants. Only one mutation, corresponding to a single amino acid change within the H-box of the kinase domain, was found in an osmotolerant strain. These results suggest that AbNIK1p participates in osmoregulation and that expression of the fully functional enzyme is essential for dicarboximide and phenylpyrrole antifungal activities.

A two-component histidine kinase of the rice blast fungus is involved in osmotic stress response and fungicide action

We isolated and characterized a histidine kinase gene ( HIK1) from the rice blast fungus, Pyricularia oryzae ( Magnaporthe grisea). The deduced amino acid sequence of HIK1 showed highest similarity (85.7%) to a hybrid-type histidine kinase, Os-1/Nik-1 of Neurospora crassa. Disruption of HIK1 caused no defect in cell growth on normal media and in pathogenicity to rice plants. The Δ hik1 strain acquired resistance to three groups of fungicides (phenylpyrroles, dicarboximides, and aromatic hydrocarbons) similar to os-1 mutants of N. crassa. The Δ hik1 strain showed increased sensitivity to high concentrations of sugars although its salt sensitivity was not elevated, suggesting that the rice blast fungus can distinguish osmostresses caused by high sugar concentrations and high salt concentrations. In contrast, os-1 mutants of N. crassa are sensitive to high concentrations of both salts and sugars. These findings suggest that P. oryzae and N. crassa partially differ in their os (osmosensitive) signal transduction pathway.

Pyrrolnitrin Interferes with Osmotic Signal Transduction in <i>Neurospora crassa</i>

Pyrrolnitrin, produced by several bacteria that are used in biological control, has an inhibitory effect on the electron transport system of respiration in Neurospora crassa. We have previously described that fludioxonil, a derivative of phenylpyrroles, affects osmotic signal transduction. Both pyrrolnitrin and fludioxonil were highly active against Botrytis cinerea, Fusarium oxysporum, Rhizoctonia solani, and N. crassa. However, a high concentration of pyrrolnitrin (more than 10 μg/ml) inhibited the growth of fludioxonil-insensitive fungi such as Pythium ultimum, Phytophthora capsici, and Saccharomyces cerevisiae. In order to clarify the difference in the antifungal mechanisms between pyrrolnitrin and fludioxonil, we observed cross-resistance in mutants of the osmotic signal transduction pathway, namely, os-1 (histidine kinase), os-4 (MAPKK kinase), os-5 (MAPK kinase), and os-2 (MAP kinase) of N. crassa. All os mutants that were resistant to fludioxonil showed cross-resistance to pyrrolnitrin without exception. The levels of resistance to pyrrolnitrin correlated well with those to fludioxonil in the 10 os-1 mutant alleles with single amino acid substitutions. However, at a concentration of 6.1 μg/ml, pyrrolnitrin inhibited the growth of all strains including the os mutants insensitive to fludioxonil even at 25 μg/ml. When the conidia of the wild-type strain were grown on a medium containing either fungicide at a concentration of 0.1 μg/ml, both fungicides induced the swelling and rupture of conidia without germ-tube formation. At a concentration of 25 μg/ml, pyrrolnitrin inhibited conidia germination without any morphological change in the fludioxonil-insensitive os-S mutant. Both fungicides at a concentration of 1 μg/ml stimulated glycerol synthesis in the wild-type strain, but the glycerol content was reduced to a considerable extent on treatment with 25 μg/ml pyrrolnitrin. These results suggest that a primary antifungal mechanism of pyrrolnitrin against N. crassa is interference with the osmotic signal transduction pathway rather than inhibition of respiration.

Fungicide Resistance and Osmotic Stress Sensitivity in os Mutants of Neurospora crassa

The os-1 (osmotic sensitive-1) mutants in Neurospora crassa were classified into two groups based on their resistance to various fungicides (e.g., fludioxonil, iprodione, and quintozene) and osmotic stress. The first group of strains, represented by NM233(t) and Y256M209, grew on medium containing fludioxonil even at a concentration of 25 μg/ml but their growth was inhibited only when a high concentration of NaCl was used for osmotic stress. The second group of strains, represented by M155-1, M16, P3282, and P5990, was less resistant to fludioxonil than the first group, but their growth was inhibited even under low osmotic stress. Both groups of the os-1 mutant strains transformed by the wild-type os-1 gene restored fungicide sensitivity and osmotic tolerance, suggesting that the os-1 gene mutations are responsible for phenotypes in both groups of os-1 mutants. The results indicated that the level of resistance to fludioxonil is not related to that of osmotic sensitivity in the os-1 mutant strains. To investigate the correlation between fungicide resistance and osmotic sensitivity in other os mutant strains, we isolated revertant strains from the os-5 mutant strain on medium containing 6% NaCl. The revertant os-5R-18 strain reversed its osmotic sensitivity to the level of the wild-type strain but still remained resistant to fludioxonil. Genetic analysis suggested that an additional mutation, named su(os-5), in the revertant os-5R-18 strain suppressed the osmotic sensitivity of the os-5 mutant strain but did not do so with those of the os-1 and os-2 mutant strains. The os-5 mutant strain accumulated less glycerol by osmotic stress, while glycerol synthesis in the revertant os-5/su(os5) strain was induced by high osmolarity to the level of the wild-type strain. However, glycerol accumulation by fludioxonil observed in the wild-type strain did not occur in either strain with os-5 and os-5/su(os5). These results suggested that phenylpyrroles affect glycerol synthesis in the osmotic signal transduction pathway, but that high osmotic sensitivity is not directly linked with resistance to dicarboximides and phenylpyrroles.

Sensitivity to Phenylpyrrole Fungicides and Abnormal Glycerol Accumulation in <i>Os</i> and <i>Cut</i> Mutant Strains of <i>Neurospora crassa</i>

アカパンカビの浸透圧感受性株に対する薬剤感受性を調べたところ, os-1, os-2, os-4, os-5変異株が, ジカルボキシイミド剤, 芳香族炭素系剤と同様にフルジオキソニルにも交差耐性を示すことを見いだした. os-2, os-4, os-5変異株は各剤に高度の耐性を示したが, os-1変異株は低度耐性でイプロジオン (LD50: 14μg/ml) とフルジオキソニル (LD50: 0.087μg/ml) に対し感受性を残していた. 一方, 浸透圧感受性 cut 変異株は野生株と同等の薬剤感受性を示した. また, 野生株をフルジオキソニル処理すると分生胞子の膨潤と破裂が誘導されたが, この形態異常は cut 変異株でも同様に観察された. フルジオキソニル, イプロジオンを野生株に処理するとグリセロールの異常蓄積が認められることが報告されているが, これらのグリセロール異常蓄積は薬剤に高度耐性を示すos-2, os-4, os-5変異株では認められなかった. また, 低度耐性os-1変異株を10μg/mlの薬剤濃度で処理した場合に, フルジオキソニルでは顕著なグリセロールの蓄積が認められたが, イプロジオンではグリセロール蓄積量は野生株と比較し低下していた. これらの結果から薬剤耐性とグリセロール異常蓄積の相関が推定された. しかし, 浸透圧感受性 cut 変異株は薬剤に野生株同等の感受性を示したにも関わらず, フルジオキソニル, イプロジオンによるグリセロールの蓄積誘導は認められなかった. 高浸透圧下では, os変異株はグリゼロールの蓄積を示したが蓄積量は野生株より低下していた. 一方, cut 変異株では高浸透圧条件下でもグリセロール蓄積は認められず, 同変異株ではグリセロール合成能が欠損していると推定された. フルジオキソニル, イプロジオンはグリセロール合成能を欠失した株に対しても野生株と同じ抗菌作用を示すと考えられることから, これらの化合物は浸透圧のシグナル伝達をかく乱するが, グリセロールの異常蓄積は直接抗菌作用発現に関与していないと考えられた.

Organelle movements in the wild type and wall-less fz;sg;os-1 mutants of Neurospora crassa are mediated by cytoplasmic microtubules.

The cellular basis of organelle transport in filamentous fungi is still unresolved. Here we have studied the intracellular movement of mitochondria and other organelles in the fungus Neurospora crassa. Four different model systems were employed: hyphae, protoplasts, a cell wallless mutant, and experimentally generated small, flattened cell fragments of the mutant cells. Organelle movements were visualized by DIC optics and computer-enhanced video microscopy. In all cell models the transport of organelles was vectorial and saltatory in nature. The mean velocities for mitochondria, particles and nuclei were 1.4, 2.0, and 0.9 microns/s, respectively. Treatment with 10 microM nocodazole for 30 minutes caused a complete disappearance of microtubules and reversibly blocked directed transport of virtually all organelles, whereas cytochalasin D up to 20 microM was without effect. Correlative video and immunofluorescence microscopy of small fragments of wall-less mutant cells revealed a clear match between microtubule distribution and the tracks of moving organelles. We conclude that organelle movement in the filamentous fungus Neurospora crassa is a microtubule-dependent process.

Effect of osmotic stress on yield and polyol content of dicarboximide-sensitive and -resistant strains of Neurospora crassa

A wild type strain and an os-1 mutant of Neurospora crassa were subjected to osmotic stress in media of high osmolality (865 mosmol kg−1) adjusted with sodium chloride, potassium chloride, glucose, fructose, mannitol or glycerol. The strains produced similar amounts of mycelia when grown in basal medium (125 mosmol kg−1) or in medium of high osmolality adjusted with glycerol. In media adjusted with other osmotica, yields of wild type and mutant mycelia were reduced by approximately 35% and 90%, respectively. Both strains accumulated large amounts of glycerol and, to a lesser extent, mannitol when grown in media of high osmolality adjusted with sodium chloride. These alditols contributed a solute potential equivalent to approximately 30–40% of the external water potential. Wild type and mutant mycelia that had been washed during harvesting with water or iso-osmotic culture medium contained much less glycerol and mannitol than equivalent unwashed mycelia. The osmotic sensitivity of the os-1 mutant is discussed in relation to osmotic adjustment and retention of glycerol.

Spindle-pole organization during early mouse development

Spindle-pole organization during early mouse development was examined using a variety of immunological reagents that recognize centrosomal components. Spindle poles of unfertilized eggs and blastocysts were found to react positively with two antisera (centrin and NRS-01), whereas poles of activated eggs and early cleavage-stage embryos were negative when treated with the same sera. In contrast, a third antiserum (5051) showed positive spindle-pole staining throughout the preimplantation stages of development. Two monoclonal antibodies (MPM-1 and MPM-2) that are known to react with mitotic phosphoproteins were also used in this study. Both antibodies stained the cytoplasm of mitotic cells with extremely high intensity. In addition, MPM-2 was found to stain spindle poles. These results suggest that organizational changes in the spindle pole are occurring during early mouse development. Embryos homozygous for a recessive lethal mutation known as oligosyndactyly ( Os) were also treated with the reagents described above. This mutation results in a metaphase arrest at the blastocyst stage with intact spindles being present. Spindle poles were observed in Os homozygous mutants stained with centrin, NRS-01, and 5051. However, when Os mutants were stained with the MPM monoclonal antibodies, about half of the mitotic cells completely lacked the dramatic cytoplasmic staining. This observation is in contrast to that observed for wild-type embryos, where greater than 95% of mitotic cells showed positive cytoplasmic staining.

Alterations in the biosynthesis of chitin and glucan in the slime mutant of Neurospora crassa

The slime mutant of Neurospora crassa lacks both chitin and insolubleβ(1 → 3)glucans either cellbound or secreted to the medium. Failure to synthesize both polymers is not due to deficiencies in the precursor sugar nucleotides, UDP-GlcNAc and UDP-Glc, since these are present in higher amounts in the mutant than in the wild type. The slime mutant also contains higher levels of chitin synthetase than the wild type, but lacks detectable glucan synthetase activity. an os-1 mutant grown in hypertonic medium contained reduced amounts of chitin and glucan, but its glucan and chitin synthetase activities were nearly normal. It is concluded that the absence of a cell wall in the slime mutant is due to a mutational event(s) which affects the mechanism of activation or delivery of chitin synthetase to the cell surface and the formation of a functionalβ(1 → 3)glucan synthetase. This mutational event must be different in the os-1 strain.

The morphology and ultrastructure of the spore and exosporium of some Clostridium species.

Surmmary The spores of original isolates of Clostridium species obtained from fresh and processed marine fish and from marine bottom deposits were compared with the spores of culture collection strains of the same species. These strains were Clostridium botulinum type E, C. sporogenes, C. bifermentans and C. sordellii. Spores of unclassified, non-toxigenic isolates which resemble C. botulinum type E, of ‘OS mutants’, of culture collection strains of C. botulinum types A and B and an isolate of type F were also examined. The gross morphology and ultrastructure of the spores and exosporia were studied in electron microscopic preparations by using the techniques of metal shadowing, carbon replica, negative-staining and ultra-thin sectioning. Spores of some strains were found to possess tubular appendages. The morphology of the spores and the ultrastructural detail of the spore appendages and exosporia are described.