Mating type loci inFusarium: structure and function

Sex in fungi is regulated by highly dissimilar mating type loci named idiomorphs. The genus Fusarium harbours both sexual as well as esexual species and each appears to contain one or the other idiomorph. The structure of these loci is highly conserved, suggesting a cryptic sexual cycle in these socalled asexual species. Alternatively, idiomorphs could regulate additional hitherto unrecognized biological processes. Such processes could be elucidated by expression profiling using mutants disrupted in their mating type loci.     

Sporisorium reilianum possesses a pool of effector proteins that modulate virulence on maize

The biotrophic maize head smut fungus Sporisorium reilianum is a close relative of the tumour-inducing maize smut fungus Ustilago maydis with a distinct disease aetiology. Maize infection with S. reilianum occurs at the seedling stage, but spores first form in inflorescences after a long endophytic growth phase. To identify S. reilianum-specific virulence effectors, we defined two gene sets by genome comparison with U. maydis and with the barley smut fungus Ustilago hordei. We tested virulence function by individual and cluster deletion analysis of 66 genes and by using a sensitive assay for virulence evaluation that considers both disease incidence (number of plants with a particular symptom) and disease severity (number and strength of symptoms displayed on any individual plant). Multiple deletion strains of S. reilianum lacking genes of either of the two sets (sr10057, sr10059, sr10079, sr10703, sr11815, sr14797 and clusters uni5-1, uni6-1, A1A2, A1, A2) were affected in virulence on the maize cultivar ‘Gaspe Flint’, but each of the individual gene deletions had only a modest impact on virulence. This indicates that the virulence of S. reilianum is determined by a complex repertoire of different effectors which each contribute incrementally to the aggressiveness of the pathogen.     

The biological cycle of Sporisorium reilianum f.sp. zeae: an overview using microscopy

Sporisorium reilianum f.sp. zeae is the causal agent of maize head smut. Using microscopy, we describe the development of the fungus during its saprophytic and parasitic phase. When compatible, the yeast forms fused to produce dicaryotic hyphae. These hyphae were infectious and penetrated the maize in the root. Surprisingly, the formation of conjugation tubes was rarely observed in vitro. In contrast, extensive development of long hyphae was observed from the haploid form of the yeast, these hyphae being able to fuse when arising from compatible strains. In planta, the fungus acted as a biotrophic endophyte until sporogenesis, which occurred in the floral meristem of the maize. The symptoms of the infection were reduced. Penetration in the root was never accompanied by drastic damage of the host cell and we did not observe thickening or apposition of plant material to reinforce the wall structure. Moreover, the fungus was embedded in an amorphous matrix and thus appeared isolated from the host cell. In the floral meristem, radical changes were observed, the host cell was totally invaded by the fungus in the course of sporogenesis. The deposits observed on the fungal wall are likely related to the echinulation of the teliospores.     

Effects of a fraction from maize root exudates on haploid strains of Sporisorium reilianum f. sp. zeae

Sporisorium reilianum f. sp. zeae is the causal agent of head smut of maize. Although the main symptom of this disease is the formation of a black fungal sorus on the reproductive parts of the maize, the infection always occurs via the roots. Early infection stages are characterised by a hyphal proliferation of the fungus around the roots. In this paper, we describe effects of a fraction extracted from maize root exudates on growth of S. reilianum f. sp. zeae. The fungus grew as a yeast form on artificial medium, but in presence of these fractions, some yeasts switched to a hyphal form. In addition, an increased proliferation of the yeast form was also observed with exudates from a variety of maize susceptible to head smut. In the presence of exudates obtained from a tolerant variety of maize, proliferation of the yeast form was inhibited, whereas the induction of yeast-hypha transition was always observed. These results indicated that some molecules in root exudates could play a role in the pre-infectious stage between maize and S. reilianum f. sp. zeae.     

子囊菌交配型位点与交配型基因研究进展

有性生殖是真菌的生殖方式之一,是真菌遗传重组的重要驱动力。交配型(mating-type,MAT)位点控制真菌性别,在有性生殖过程中起决定性作用。不同类型真菌MAT位点的基因组成、排列方式和编码蛋白不尽相同。近年来,MAT位点和MAT基因的功能与调控网络研究进展较快。本文对子囊菌交配型位点的基因组成及分布、MAT基因的功能、MAT位点与有性生殖调控通路的关系等进行了综述。     

Effect of proteolytic and glycolytic enzymes on a factor inSorghum bicolor that induces mycelial growth in the smut fungus,Sporisorium reilianum

Proteins obtained from seedling shoots and floral meristems ofSorghum bicolor (L.) Moench cv. NK 1210 induced mycelial growth in the smut fungus,Sporisorium reilianum in vitro. Proteins precipitated with trichloroacetic acid and ammonium sulfate were equally effective as inducers, although there were minor variations in the pattern of mycelial growth. Hydrolysis of the protein fraction with the proteolytic enzyme pronase E resulted in considerable reduction in the proteins' ability to induce mycelial growth. Digestion of the protein fraction with driselase, resulted in a slight enhancement of biological activity. The results suggest that amino sugar moieties in glycoproteins may act as inducers of mycelial growth inSporisorium reilianum.     

Cyborg lectins: novel leguminous lectins with unique specificities

Bauhinia purpurea lectin (BPA) is one of the beta-galactose-binding leguminous lectins. Leguminous lectins contain a long metal-binding loop, part of which determines their carbohydrate-binding specificities. Random mutations were introduced into a portion of the cDNA coding BPA that corresponds to the carbohydrate-binding loop of the lectin. An library of the mutant lectin expressed on the surface of lambda foo phages was screened by the panning method. Several phage clones with an affinity for mannose or N-acetylglucosamine were isolated. These results indicate the possibility of making artificial lectins (so-called "cyborg lectins") with distinct and desired carbohydrate-binding specificities.     

Identification of a New Race of Sporisorium reilianum and Characterization of the Reaction of Sorghum Lines to Four Races of the Head Smut Pathogen

Sporisorium reilianum is the causal agent of head smut on sorghum and maize. In order to effectively utilize host resistance to control this important disease in crops, it is necessary to monitor changes in disease dynamics and virulence of the pathogen. An outbreak of head smut was recently observed in a sorghum field, near Gaoping, Shanxi, China, and research was undertaken to characterize a putative new race of S. reilianum. A set of differential sorghum lines with resistance to several conventional races was used to characterize the newly collected isolate of S. reilianum. The reactions of differential cultivars/germplasm lines to the new isolate indicate that it is a new physiological race of S. reilianum. The new race is highly virulent on sorghum line A₂V4 and its hybrid, Jinza 12, that are known as resistant to all existing Chinese races of S. reilianum, including races 1, 2, and 3. The new isolate of S. reilianum is different from all of the described races of the pathogen; thus, it is designated as race 4 of S. reilianum. Furthermore, a collection of 34 sorghum genotypes including commercial cultivars and germplasm lines was evaluated for disease reaction to the newly described race and the three known races of the pathogen.     

Reaction pattern of a novel thermostable α-amylase

A novel thermostable α-amylase, D45 was studied for its reaction pattern on starch hydrolysis. Fine structures of the dextrins and oligosaccharides produced by D45 were determined and compared with those produced by other thermostable α-amylases, Termamyl^®LC (LC) and Termamyl^®SC (SC). Waxy maize starch dispersion was hydrolyzed with LC, SC and D45 at different concentrations to obtain hydrolysates with the same dextrose equivalent value (DE). At DE 13, molecular weight distribution of dextrins produced by D45 displayed a mono-distribution with a peak centered at degree of polymerization (DP) of 7, whereas LC and SC hydrolysates displayed a bimodal-distribution of the molecular weight profiles with one peak centered at DP 5 and the other at DP 34. Thin-layer chromatograms (TLC) showed that DP 2, 3, 5, 6 and 7 were the primary oligosaccharides produced in LC hydrolysate, DP 4–7 in SC hydrolysate, and DP 6–9 in D45 hydrolysate. Comparison of the decrease in the blue color of amylose-iodine complex at 620 nm (blue value) with the increase in reducing value for the hydrolysis of amylose by LC, SC and D45 showed that for an equivalent decrease in blue value, LC and SC produced a higher percentage of reducing sugar than did D45. The results suggest that D45 has a greater degree of random attack (multichain) reaction, whereas LC and SC have more multiple-attack reactions.     

Diversity of N-acetylglucosamine-6-O-sulfotransferases: molecular cloning of a novel enzyme with different distribution and specificities

N-Acetylglucosamine-6-O-sulfotransferase (GlcNAc6ST) transfers sulfate to the C-6 position of non-reducing N-acetylglucosamine (GlcNAc) residues. We cloned human and mouse cDNAs encoding a novel GlcNAc6ST, designated as GlcNAc6ST-4, which showed sequence identities of 26 to 41% to other GlcNAc6STs. Human organs with strong expression of the enzyme mRNA were the heart, spleen, and ovary, while in the mouse strong expression was detected in the kidney. The enzyme expressed in CHO cells preferentially acted on mannose-linked GlcNAc, while a core 2 mucin-type oligosaccharide and an N-acetyllactosamine oligomer also served as acceptors. The distribution and the specificity of GlcNAc6ST are different from those of GlcNAc6STs identified previously.     

Identification of three mutant loci conferring carboxin-resistance and development of a novel transformation system in Aspergillus oryzae

Mutants exhibiting resistance to the fungicide, carboxin, were isolated from Aspergillus oryzae, and the mutations in the three gene loci, which encode succinate dehydrogenase (SDH) B, C, and D subunits, were identified to be independently responsible for the resistance. A structural model of the SDH revealed the different mechanisms that confer carboxin-resistance in different mutations. The mutant AosdhB gene (AosdhB(cxr)) was further examined for possible use as a transformant selection marker. After transformation with AosdhB(cxr), carboxin-resistant colonies appeared within 4 days of culture, and all of the examined colonies carried the transgene. Insertion analyses revealed that the AosdhB(cxr) gene was integrated into AosdhB locus via homologous recombination at high efficiency. Furthermore, AosdhB(cxr) functioned as a successful selection marker in a transformation experiment in Aspergillus parasiticus, suggesting that this transformation system can be used for Aspergillus species.     

Physical separation of aortic corticoid receptors with type I and type II specificities

Previous gel filtration binding assay studies indicated that rat vascular smooth muscle cells contained corticoid receptor I and corticoid receptor II sites which could be distinguished on the basis of their relative affinities for aldosterone and dexamethasone. Ion-exchange chromatography experiments were designed to separate the two sites for further studies on their physical characteristics and role in vascular smooth muscle cell physiology. Cultured aortic cells were incubated with 5-10 nM 3H steroid alone or in the presence of 10-fold non-radioactive steroid competitor for 30 min at 37 degrees C. Following cell lysis, total cellular protein-bound steroid was isolated using Sephadex G-25 and applied to a DEAE-cellulose ion-exchange column. Three peaks of radioactivity were eluted using a 1-200 mM sodium phosphate gradient: peak I (30-38 mM), peak II (52-64 mM), and peak III (92-102 mM). Peaks I and II contained 60% of the eluted radioactivity and exhibited the same steroid specificity as corticoid receptor II sites (dexamethasone greater than aldosterone). Peak III contained 40% of the eluted radioactivity and exhibited the same steroid specificity as corticoid receptor I sites (aldosterone greater than dexamethasone). These studies support the binding assay data on steroid specificity and relative proportion of type I and II sites. They also document the existence of type I and II corticoid receptors with different physicochemical characteristics in rat aortic smooth muscle cells.     

Exclusion of linkage of loci on chromosome 19 with multiple endocrine neoplasia, type 2

Linkage between seven loci on chromosome 19 and multiple endocrine neoplasia type 2a (MEN2A) was examined in a single large Swedish pedigree. A total of 50 cM was excluded from the male genetic map by pairwise analysis and an estimated 63 cM by multipoint analysis. Using existing data on the likelihood of different marker-marker distances and taking into account current exclusions on other chromosomes, the probability that the gene for MEN2A segregating in this pedigree could still be located on chromosome 19 is approximately 0.28%.     

Genetic structure of Mexican Mestizos with type 2 diabetes mellitus based on three STR loci

Background

The aims of this population genetics study were: 1) to ascertain whether Mexicans with type 2 diabetes mellitus (DM) were genetically homogeneous and 2) to compare the genetic structure of this selected population with the previously reported data of four random populations (Nuevo León, Hispanics, Chihuahua, and Central Region of Mexico).

Methods

A sample of 103 unrelated individuals with DM and whose 4 grandparents were born in five zones of Mexico was interviewed in 32 Medical Units in the Mexican Institute of Social Security (IMSS). The non-coding STRs D16S539, D7S820, and D13S317 were analyzed.

Results

Genotype distribution was in agreement with Hardy–Weinberg expectations for all three markers. Allele frequencies were found to be similar between the selected population and the four random populations. Gene diversity analysis suggested that more than 99.57% of the total gene diversity could be attributed to variation between individuals within the population and 0.43% between the populations.

Conclusions

According to the present and previous studies using molecular and non-molecular nuclear DNA markers not associated with any disease, the Mexican Mestizo population is found to be genetically homogeneous and therefore the genetic causes of DM are less heterogeneous, thereby simplifying genetic epidemiological studies as has been found in a previous study with the same design in Mexican women with breast cancer.     

Analysis ofH-2 mutants: Evidence for multiple CML target specificities controlled by theH-2K b gene

C57BL/6 (H-2 ^b ) mice and two mutants derived from this strain, B6.C-H-2 ^ba (Hz1) andE6-H-2 ^bd (M505), were studied in a number of functional tests, in vitro and in vivo, that assay for differences at theH-2 complex. All three strains give rise to reciprocal mixed lymphocyte reactivity (MLR) and cell-mediated lympholysis (CML) in vitro as well as graft-host reactivity (GVHR) and skin graft rejection in vivo. Analysis for cross-reactivity between these strains in CML revealed that the gained antigens in each mutant do not cross-react, and that Hz1 has lost an antigen shared by C57BL/6 and M505 strains. In addition, spleen cells from B10.A(4R) mice, which differ from theH-2 ^b haplotype only at theK end of theH-2 complex, recognize a common antigen shared by all three strains tested. Provided that the mutations occurred in theH-2K ^b gene, these data indicate that a) there are at least three antigenic specificities coded for by theH-2K ^b gene(s) that serve as targets for receptors on thymus-derived (T) cells in CML; b) since C57BL/6 strain mice and the mutants are serologically indistinguishable on a qualitative basis, the antigens recognized by the receptors on T cells and by humoral H-2 antibody are nonidentical; and c) mutation in theH-2K ^b locus itself can give rise to allogeneic recognition phenomena such as MLR and GVHR.     

Ribosomal RNA genes of Neurospora crassa: multiple copies and specificities

Ribosomal RNA genes were isolated from the germinated conidial and mycelial cells of N. crassa by repeated cycles of 3H-DNA:rRNA reactions followed by hydroxyapatite chromatography. Specificity of multiple copies of those rDNAs with respect to N. crassa cell types was studied. The fraction of N. crassa germinated conidial in vitro labelled 3H-DNA recovered in the presence of rRNA isolated from the same cell type was about 2.2%, when compared with approximately 1.2% rDNAs obtained in mycelial cells. These isolated rDNAs reacted specifically to 26S and 17S rRNAs of eukaryotic (N. crassa) organisms and did not react with 4S tRNAs. rRNA:rDNA reassociation kinetics studies indicate that 90% of the rRNA genes were homogeneous and not identical with the other 10% rRNA genes isolated from N. crassa mycelia. These studies suggest that the possible heterogeneity of rDNA sequences of N. crassa cannot be attributed to inclusion of any tDNA sequences as has been shown in the heterogeneity of rDNA sequences of the bacterium Escherichia coli. The heterogeneity of multiple copies of N. crassa rDNAs could be due to differences in internal or external spacer regions of N. crassa rRNA genes.