Fully Deacetylated Chitooligosaccharides Act as Efficient Glycoside Hydrolase Family 18 Chitinase Inhibitors

Small molecule inhibitors against chitinases have potential applications as pesticides, fungicides, and antiasthmatics. Here, we report that a series of fully deacetylated chitooligosaccharides (GlcN)_2–7 can act as inhibitors against the insect chitinase OfChtI, the human chitinase HsCht, and the bacterial chitinases SmChiA and SmChiB with IC₅₀ values at micromolar to millimolar levels. The injection of mixed (GlcN)_2–7 into the fifth instar larvae of the insect Ostrinia furnacalis resulted in 85% of the larvae being arrested at the larval stage and death after 10 days, also suggesting that (GlcN)_2–7 might inhibit OfChtI in vivo. Crystal structures of the catalytic domain of OfChtI (OfChtI-CAD) complexed with (GlcN)_5,6 were obtained at resolutions of 2.0 Å. These structures, together with mutagenesis and thermodynamic analysis, suggested that the inhibition was strongly related to the interaction between the −1 GlcN residue of the inhibitor and the catalytic Glu¹⁴⁸ of the enzyme. Structure-based comparison showed that the fully deacetylated chitooligosaccharides mimic the substrate chitooligosaccharides by binding to the active cleft. This work first reports the inhibitory activity and proposed inhibitory mechanism of fully deacetylated chitooligosaccharides. Because the fully deacetylated chitooligosaccharides can be easily derived from chitin, one of the most abundant materials in nature, this work also provides a platform for developing eco-friendly inhibitors against chitinases.     

A High-Throughput Gene Disruption Methodology for the Entomopathogenic Fungus Metarhizium robertsii

Systematic gene disruption is a direct way to interrogate a fungal genome to functionally characterize the full suite of genes involved in various biological processes. Metarhizium robertsii is extraordinarily versatile, and it is a pathogen of arthropods, a saprophyte and a beneficial colonizer of rhizospheres. Thus, M. robertsii can be used as a representative to simultaneously study several major lifestyles that are not shared by the “model” fungi Saccharomyces cerevisiae and Neurospora crassa; a systematic genetic analysis of M. robertsii will benefit studies in other fungi. In order to systematically disrupt genes in M. robertsii, we developed a high-throughput gene disruption methodology, which includes two technologies. One is the modified OSCAR-based, high-throughput construction of gene disruption plasmids. This technology involves two donor plasmids (pA-Bar-OSCAR with the herbicide resistance genes Bar and pA-Sur-OSCAR with another herbicide resistance gene Sur) and a recipient binary plasmid pPK2-OSCAR-GFP that was produced by replacing the Bar cassette in pPK2-bar-GFP with a ccdB cassette and recombination recognition sites. Using this technology, a gene disruption plasmid can be constructed in one cloning step in two days. The other is a highly efficient gene disruption technology based on homologous recombination using a Ku70 deletion mutant (ΔMrKu70) as the recipient strain. The deletion of MrKu70, a gene encoding a key component involved in nonhomologous end-joining DNA repair in fungi, dramatically increases the gene disruption efficiency. The frequency of disrupting the conidiation-associated gene Cag8 in ΔMrKu70 was 93% compared to 7% in the wild-type strain. Since ΔMrKu70 is not different from the wild-type strain in development, pathogenicity and tolerance to various abiotic stresses, it can be used as a recipient strain for a systematic gene disruption project to characterize the whole suite of genes involved in the biological processes of M. robertsii.     

Characterization of Glycosynthase Mutants Derived from Glycoside Hydrolase Family 10 Xylanases

Four xylanases belonging to glycoside hydrolase family 10—Thermotoga maritima XylB (TM), Clostridium stercorarium XynB (CS), Bacillus halodurans XynA (BH), and Cellulomonas fimi Cex (CF)—were converted to glycosynthases by substituting the nucleophilic glutamic acid residues with glycine, alanine, and serine. The glycine mutants exhibited the highest levels of glycosynthase activity with all four enzymes. All the glycine mutants formed polymeric β-1,4-linked xylopyranose as a precipitate during reaction with α-xylobiosyl fluoride. Two glycine mutants (TM and CF) recognized X₂ as an effective acceptor molecule to prohibit the formation of the polymer, while the other two (CS and BH) did not. The difference in acceptor specificity is considered to reflect the difference in substrate affinity at their +2 subsites. The results agreed with the structural predictions of the subsite, where TM and CF exhibit high affinity at subsite 2, suggesting that the glycosynthase technique is useful for investigating the affinity of +subsites.     

Structural,Biochemical, and Computational Characterization of the Glycoside Hydrolase Family 7 Cellobiohydrolase of the Tree-killing Fungus Heterobasidion irregulare

Root rot fungi of the Heterobasidion annosum complex are the most damaging pathogens in temperate forests, and the recently sequenced Heterobasidion irregulare genome revealed over 280 carbohydrate-active enzymes. Here, H. irregulare was grown on biomass, and the most abundant protein in the culture filtrate was identified as the only family 7 glycoside hydrolase in the genome, which consists of a single catalytic domain, lacking a linker and carbohydrate-binding module. The enzyme, HirCel7A, was characterized biochemically to determine the optimal conditions for activity. HirCel7A was crystallized and the structure, refined at 1.7 Å resolution, confirms that HirCel7A is a cellobiohydrolase rather than an endoglucanase, with a cellulose-binding tunnel that is more closed than Phanerochaete chrysosporium Cel7D and more open than Hypocrea jecorina Cel7A, suggesting intermediate enzyme properties. Molecular simulations were conducted to ascertain differences in enzyme-ligand interactions, ligand solvation, and loop flexibility between the family 7 glycoside hydrolase cellobiohydrolases from H. irregulare, H. jecorina, and P. chrysosporium. The structural comparisons and simulations suggest significant differences in enzyme-ligand interactions at the tunnel entrance in the −7 to −4 binding sites and suggest that a tyrosine residue at the tunnel entrance of HirCel7A may serve as an additional ligand-binding site. Additionally, the loops over the active site in H. jecorina Cel7A are more closed than loops in the other two enzymes, which has implications for the degree of processivity, endo-initiation, and substrate dissociation. Overall, this study highlights molecular level features important to understanding this biologically and industrially important family of glycoside hydrolases.     

Purification and Characterization of an Aspecific Glycoside Hydrolase from the Anaerobic Ruminal Fungus Neocallimastix frontalis

A glycoside hydrolase characterized by beta-fucosidase (EC 3.2.1.38) and beta-glucosidase (EC 3.2.1.21) activities was purified from the culture medium of the anaerobic ruminal phycomycete Neocallimastix frontalis grown on 0.5% Avicel. The enzyme had a molecular mass of 120 kilodaltons and a pI of 3.85. Optimal activity against p-nitrophenyl-beta-d-fucoside and p-nitrophenyl-beta-D-glucoside occurred at pH 6.0 and 50 degrees C. The beta-fucosidase and beta-glucosidase activities were stable from pH 6.0 to pH 7.8 and up to 40 degrees C. They were both inhibited by gluconolactone, sodium dodecyl sulfate, p-chloromercuribenzoate, and Hg cation. The enzyme had K(m)s of 0.26 mg/ml for p-nitrophenyl-beta-d-fucoside and 0.08 mg/ml for p-nitrophenyl-beta-d-glucoside. The purified protein also had low beta-galactosidase activity.     

Impact of a Nematode-parasitic Fungus on the Effectiveness of Entomopathogenic Nematodes

The impact of the nematode-parasitic fungus Hirsutella rhossiliensis on the effectiveness of Steinernema carpocapsae, S. glaseri, and Heterorhabditis bacteriophora against Galleria mellonella larvae was assessed in the laboratory. The presence of Hirsutella conidia on the third-stage (J3) cuticle of S. carpocapsae and H. bacteriophora interfered with infection of insect larvae. Conidia on the J3 cuticle of S. glaseri and on the ensheathing second-stage cuticle of H. bacteriophora did not reduce the nematodes'' ability to infect larvae. The LD₅₀ values for S. carpocapsae, S. glaseri, and H. bacteriophora in sand containing H. rhossiliensis were not different from those in sterilized sand when Galleria larvae were added at the same time as the nematodes. However, when Galleria larvae were added 3 days after the nematodes, the LD₅₀ of S. glaseri was higher in Hirsutella-infested sand than in sterilized sand, whereas the LD₅₀ of H. bacteriophora was the same in infested and sterilized sand. Although the LD₅₀ of S. carpocapsae was much higher in Hirsutella-infested sand than in sterilized sand, the data were too variable to detect a significant difference. These data suggest that H. bacteriophora may be more effective than Steinernema species at reducing insect pests in habitats with abundant nematode-parasitic fungi.     

Sequence,Structure, and Evolution of Cellulases in Glycoside Hydrolase Family 48

Currently, the cost of cellulase enzymes remains a key economic impediment to commercialization of biofuels (1). Enzymes from glycoside hydrolase family 48 (GH48) are a critical component of numerous natural lignocellulose-degrading systems. Although computational mining of large genomic data sets is a promising new approach for identifying novel cellulolytic activities, current computational methods are unable to distinguish between cellulases and enzymes with different substrate specificities that belong to the same protein family. We show that by using a robust computational approach supported by experimental studies, cellulases and non-cellulases can be effectively identified within a given protein family. Phylogenetic analysis of GH48 showed non-monophyletic distribution, an indication of horizontal gene transfer. Enzymatic function of GH48 proteins coded by horizontally transferred genes was verified experimentally, which confirmed that these proteins are cellulases. Computational and structural studies of GH48 enzymes identified structural elements that define cellulases and can be used to computationally distinguish them from non-cellulases. We propose that the structural element that can be used for in silico discrimination between cellulases and non-cellulases belonging to GH48 is an ω-loop located on the surface of the molecule and characterized by highly conserved rare amino acids. These markers were used to screen metagenomics data for “true” cellulases.     

Tertiary Structure and Characterization of a Glycoside Hydrolase Family 44 Endoglucanase from Clostridium acetobutylicum

A gene encoding a glycoside hydrolase family 44 (GH44) protein from Clostridium acetobutylicum ATCC 824 was synthesized and transformed into Escherichia coli. The previously uncharacterized protein was expressed with a C-terminal His tag and purified by nickel-nitrilotriacetic acid affinity chromatography. Crystallization and X-ray diffraction to a 2.2-Å resolution revealed a triose phosphate isomerase (TIM) barrel-like structure with additional Greek key and β-sandwich folds, similar to other GH44 crystal structures. The enzyme hydrolyzes cellotetraose and larger cellooligosaccharides, yielding an unbalanced product distribution, including some glucose. It attacks carboxymethylcellulose and xylan at approximately the same rates. Its activity on carboxymethylcellulose is much higher than that of the isolated C. acetobutylicum cellulosome. It also extensively converts lichenan to oligosaccharides of intermediate size and attacks Avicel to a limited extent. The enzyme has an optimal temperature in a 10-min assay of 55°C and an optimal pH of 5.0.Thirteen glycoside hydrolase (GH) families, each having members related to each other by amino acid sequence, contain enzymes that hydrolyze cellulose and/or cellooligosaccharides (4; http://www.cazy.org). Among them is GH family 44 (GH44), most of whose enzymes are endoglucanases (EGs). In general, EGs are more active on longer rather than on shorter chains and are more likely to attack bonds in the interiors of carbohydrate chains than those near their termini.With one exception, GH44 enzymes are produced by bacteria, both aerobic and anaerobic. At present, 29 amino acid sequences of GH44 members have been determined (4). Often they are combined with other GHs in multienzyme proteins (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Structural organization of genes coding for GH44 CDs, excluding GH44 members with only a signal peptide and a CD. The gene encoding O. terrae''s GH44 member produces a 920-residue protein whose domain structure is unclassified. The sequence was searched against the Pfam library, and a low E-value, 1.22 × 10⁻²², was found for a PKD (polycystic kidney disease)-type domain. Ig, immunoglobulin.Not all of these GH44 enzymes have been produced in vitro, and those that have been produced have only been partially characterized. Experimental results indicate that GH44 enzymes exclusively cleave β-1,4 bonds between glucosyl and xylosyl residues and that they have different abilities to attack xylan, lichenan, and different cellulose forms, such as Avicel, acid-swollen cellulose, and carboxymethyl cellulose (CMC), with the presence of a carbohydrate-binding module (CBM) allowing higher activity on solid cellulose. They appear to be inactive on short oligosaccharides, like p-nitrophenyl (PNP)-β-glucopyranoside, PNP-β-cellobioside, and PNP-β-xylopyranoside.Most GH families containing cellulases have at least one member with a known tertiary structure. That was not true of GH44 until Kitago et al. (15) published six different crystal structures of an EG, CelJ, from Clostridium thermocellum. Three of the crystal structures are of the wild-type enzyme, and the other three are of the E186Q mutant, with each form being both unliganded and complexed with cellopentaose or cellohexaose. The enzyme uses a retaining mechanism, with Glu186 being the proton donor/acceptor and Glu359 being the nucleophile. Subsites −4 to −1 of the wild-type enzyme hold cellotetraose. When the E186Q mutant is soaked with cellopentaose or cellohexaose, different-length cellooligosaccharides are complexed in its subsites −4 to +5.A second tertiary structure from an unidentified bacterium is similar to that from C. thermocellum (23). The enzyme, CelM2, is a triose phosphate isomerase (TIM)-like (β,α)₈ barrel with a β-sandwich domain. It also has Glu221 and Glu393 as the catalytic proton donor/acceptor and nucleophile, respectively. These two residues are located approximately 4 Å apart from one another, similar to the catalytic residues of CelJ.The present work concerns the GH44 putative EG from Clostridium acetobutylicum ATCC 824, a Gram-positive, mesophilic, anaerobic, solvent-producing bacterium. This organism and other solvent-producing Clostridium strains cannot grow on cellulose as a sole carbon source, but the first can produce EGs, mainly extracellular, when grown on glucose, xylose, mannose, and cellobiose (18). Nearly all of the same strains can grow on larch wood xylan, but C. acetobutylicum ATCC 824 can do this only when cultured in a chemostat, where it produces xylanase activity (19).Genomic sequencing has found the gene CAC0915, which putatively encodes a fusion protein consisting of a signal peptide, a GH44 catalytic domain (CD), and a type I dockerin but no CBM in C. acetobutylicum ATCC 824 (25). This putative protein, CAC0915, has 606 amino acids for a calculated molecular mass of 66.8 kDa (25). The same project found genes for many other cellulases and xylanases. In fact, the complete coding for a cellulosome similar to those in the cellulolytic species Clostridium cellulovorans and Clostridium cellulolyticum appears to be present in C. acetobutylicum ATCC 824 (25), and a cellulosome is produced, but its cellulolytic activity is very low (28). Schwarz et al. (29) have hypothesized that C. acetobutylicum has repressed cellulosome expression and cellulolytic activity during evolution since it can grow on simpler substrates, including starch, oligosaccharides, and monosaccharides.This article reports the phylogenetic tree of the GH44 enzymes and the production, purification, and subsequent structural and kinetic characterization of C. acetobutylicum GH44 EG. This protein apparently had not been observed in isolated form before this project.     

Crystal Structure and Mutational Analysis of Isomalto-dextranase,a Member of Glycoside Hydrolase Family 27

Arthrobacter globiformis T6 isomalto-dextranase (AgIMD) is an enzyme that liberates isomaltose from the non-reducing end of a polymer of glucose, dextran. AgIMD is classified as a member of the glycoside hydrolase family (GH) 27, which comprises mainly α-galactosidases and α-N-acetylgalactosaminidases, whereas AgIMD does not show α-galactosidase or α-N-acetylgalactosaminidase activities. Here, we determined the crystal structure of AgIMD. AgIMD consists of the following three domains: A, C, and D. Domains A and C are identified as a (β/α)₈-barrel catalytic domain and an antiparallel β-structure, respectively, both of which are commonly found in GH27 enzymes. However, domain A of AgIMD has subdomain B, loop-1, and loop-2, all of which are not found in GH27 human α-galactosidase. AgIMD in a complex with trisaccharide panose shows that Asp-207, a residue in loop-1, is involved in subsite +1. Kinetic parameters of the wild-type and mutant enzymes for the small synthetic saccharide p-nitrophenyl α-isomaltoside and the polysaccharide dextran were compared, showing that Asp-207 is important for the catalysis of dextran. Domain D is classified as carbohydrate-binding module (CBM) 35, and an isomaltose molecule is seen in this domain in the AgIMD-isomaltose complex. Domain D is highly homologous to CBM35 domains found in GH31 and GH66 enzymes. The results here indicate that some features found in GH13, -31, and -66 enzymes, such as subdomain B, residues at the subsite +1, and the CBM35 domain, are also observed in the GH27 enzyme AgIMD and thus provide insights into the evolutionary relationships among GH13, -27, -31, -36, and -66 enzymes.     

The Mosquito Melanization Response Is Implicated in Defense against the Entomopathogenic Fungus Beauveria bassiana

Mosquito immunity studies have focused mainly on characterizing immune effector mechanisms elicited against parasites, bacteria and more recently, viruses. However, those elicited against entomopathogenic fungi remain poorly understood, despite the ubiquitous nature of these microorganisms and their unique invasion route that bypasses the midgut epithelium, an important immune tissue and physical barrier. Here, we used the malaria vector Anopheles gambiae as a model to investigate the role of melanization, a potent immune effector mechanism of arthropods, in mosquito defense against the entomopathogenic fungus Beauveria bassiana, using in vivo functional genetic analysis and confocal microscopy. The temporal monitoring of fungal growth in mosquitoes injected with B. bassiana conidia showed that melanin eventually formed on all stages, including conidia, germ tubes and hyphae, except the single cell hyphal bodies. Nevertheless, melanin rarely aborted the growth of any of these stages and the mycelium continued growing despite being melanized. Silencing TEP1 and CLIPA8, key positive regulators of Plasmodium and bacterial melanization in A. gambiae, abolished completely melanin formation on hyphae but not on germinating conidia or germ tubes. The detection of a layer of hemocytes surrounding germinating conidia but not hyphae suggested that melanization of early fungal stages is cell-mediated while that of late stages is a humoral response dependent on TEP1 and CLIPA8. Microscopic analysis revealed specific association of TEP1 with surfaces of hyphae and the requirement of both, TEP1 and CLIPA8, for recruiting phenoloxidase to these surfaces. Finally, fungal proliferation was more rapid in TEP1 and CLIPA8 knockdown mosquitoes which exhibited increased sensitivity to natural B. bassiana infections than controls. In sum, the mosquito melanization response retards significantly B. bassiana growth and dissemination, a finding that may be exploited to design transgenic fungi with more potent bio-control activities against mosquitoes.     

Field Trial with the Entomopathogenic Fungus Metarhizium anisopliae var. acridum against Bands of the Grasshopper Rhammatocerus schistocercoides in Brazil

The efficacy of a mycoinsecticide formulated in vegetable oil was tested in Brazil against the grasshopper Rhammatocerus schistocercoides . A set of experiments was conducted in the Chapada dos Parecis region (Mato Grosso state), a permanent zone of outbreaks for this pest. Experiments were performed in zones of natural vegetation, against grasshopper bands in the third nymphal instar. Three nymphal bands were treated with a mycoinsecticide formulation based on conidia of the entomopathogenic fungus Metarhizium anisopliae var. acridum ( =M. flavoviride ), strain CG 423. Three non-treated bands were used as control. The application was made with the aid of a hand-held ULV sprayer adjusted to deliver 2 l of the formulation ha -1 , each containing 1 ×10 13 conidia. Treatments were limited to the surface of the grasshopper bands and their immediate borders (5-10 m). The efficacy of the mycoinsecticide was evaluated through band survival after treatment (grasshopper numbers, surface, density, behaviour and daily movement of the band), allowing the insects to move freely in their natural environment. Insects were regularly surveyed and maintained in the laboratory, allowing estimates of the infection rate. Field and laboratory studies showed a clear effect of the product 10 days after treatment. At 14 days post-spraying, mortality caused by the mycoinsecticide in the field was approximately 88%.     

Crystal Structure of the Salmonella enterica Serovar Typhimurium Virulence Factor SrfJ,a Glycoside Hydrolase Family Enzyme

To cause infection, Salmonella enterica serovar Typhimurium uses type III secretion systems, which are encoded on two Salmonella pathogenicity islands, SPI-1 and SPI-2, the latter of which is thought to play a crucial role in bacterial proliferation in Salmonella-containing vacuoles (SCVs) after invading cells. S. Typhimurium SrfJ, located outside SPI-2, is also known to be involved in Salmonella pathogenicity and has high amino acid sequence homology with human lysosomal glucosylceramidase (GlcCerase). We present the first crystal structure of SrfJ at a resolution of 1.8 Å. The overall fold of SrfJ shares high structure similarities with that of human GlcCerase, comprising two distinctive domains: a (β/α)₈-barrel catalytic domain and a β-sandwich domain. As in human GlcCerase, the pocket-shaped active site of SrfJ is located on the C-terminal side of the barrel, and two conserved glutamic acid residues are used for the enzyme catalysis. Moreover, a glycerol-bound form of SrfJ reveals that the glucose ring moiety of the substrate might similarly bind to the enzyme as to human GlcCerase, suggesting that SrfJ might function as a glycoside hydrolase. Although some structural differences are observed between SrfJ and human GlcCerase in the substrate entrance of the active site, we speculate that, based on the high structural similarities to human GlcCerase in the overall fold and the active-site environment, SrfJ might have a GlcCerase activity and use the activity to enhance Salmonella virulence by modifying SCV membrane lipids.Gram-negative bacterial pathogens deliver effector proteins into host cells through type III secretion systems (TTSS). The TTSS apparatus is a molecular syringe which spans the inner and outer membranes of pathogens and secretes translocon and effector proteins. Translocon proteins locate at the tip of the needle structure and are involved in the translocation of effector proteins by forming pores in the host cell membrane (3). The translocated effector proteins function to manipulate diverse host cellular processes such as cytoskeleton assembly, vesicle transport, and signal transduction, thereby promoting bacterial virulence (9).Salmonella enteric serovar Typhimurium (S. Typhimurium) causes a systemic infection in mice and is an intensively studied model of typhoid fever. This gram-negative bacterium can invade host cells and then survive by replicating within a membrane-bound compartment known as the Salmonella-containing vacuole (SCV) (16). Both invasion and intracellular survival are mediated by numerous virulence genes, which are clustered within the pathogenicity islands, SPI-1 and SPI-2 (18). The regulation of virulence proteins encoded by each pathogenicity island depends on the different stages of infection. While most of the genes within SPI-1 are required for the invading host cells and the early stages of SCV development (8), the genes in SPI-2 play a crucial role in bacterial proliferation in SCVs after cell invasion (23). The SsrA-SsrB two-component regulatory system is known to regulate the expression of genes within SPI-2 for bacterial virulence (4). Recent works have shown that several genes located outside of SPI-2 are under the control of the SsrA-SsrB regulator as well, and these have been proposed as putative virulence factors (10, 25).S. Typhimurium SrfJ was initially identified as a gene that is strongly activated by SsrB outside SPI-2 (25). Furthermore, a mutation on srfJ leads to mild attenuation of virulence in mice (22). Interestingly, SrfJ shares high amino acid sequence similarity with human lysosomal glucosylceramidase (GlcCerase) (25), which is a peripheral membrane protein catalyzing the hydrolysis of glucosylceramide (GlcCer) to β-glucose and ceramide in the presence of the modulator protein saposin C and lipid (11). Inherited defects in GlcCerase result in lysosomal GlcCer accumulation and, as a consequence, Gaucher disease, the most common lysosomal storage disease (19). Both human GlcCerase and SrfJ have been grouped into glycoside hydrolase (GH) family 30 containing GlcCerase (EC 3.2.1.45), β-1,6-glucanse (EC 3.2.1.75), and β-xylosidase (EC 3.2.1.37) of the GH-A clan in the CAZy database (http://afmb.cnrs-mrs.fr/CAZY). Among the members of GH family 30, structural information is available only on the human enzyme. The biochemical function of SrfJ and its role in Salmonella virulence remain to be elucidated. In order to better understand the function of SrfJ, we have determined the crystal structure of SrfJ from S. Typhimurium at a resolution of 1.8 Å.     

Transcriptional and Histological Analyses of the Thymic Developmental Process in the Fetal Pig

The humanized pig model, in which human cells or tissues can be functionally maintained in pigs, can be an invaluable tool for human medical research. Although the recent development of immunodeficient pigs has opened the door for the development of such a model, the efficient engraftment and differentiation of human cells may be difficult to achieve. The transplantation of human cells into fetal pigs, whose immune system is immature, will ameliorate this problem. Therefore, we examined the development of porcine fetal thymus, which is critical for the establishment of the immune system. We first analyzed the levels of mRNA expression of genes that are relevant to the function of thymic epithelial cells or thymocytes in whole thymi from 35 to 85 days of gestation (DG) and at 2 days postpartum (DP) by quantitative RT-PCR. In addition, immunohistochemical analyses of thymic epithelial cells from DG35 to DG55 and DP2 were performed. These analyses showed that the thymic cortex was formed as early as DG35, and thymic medulla gradually developed from DG45 to DG55. These findings suggested that, at least before DG45, the thymus do not differentiate to form fully functional T cells.     

Isolation of the Mating-Type Genes of the Phytopathogenic Fungus Magnaporthe Grisea Using Genomic Subtraction

Using genomic subtraction, we isolated the mating-type genes (Mat1-1 and Mat1-2) of the rice blast fungus, Magnaporthe grisea. Transformation of M. grisea strains of one mating type with a linearized cosmid clone carrying the opposite mating-type gene resulted in many ``dual maters,' strains that contain both mating-type genes and successfully mate with both Mat1-1 and Mat1-2 testers. Dual maters differed in the frequency of production of perithecia in pure culture. Ascospores isolated from these homothallic crosses were either Mat1-1 or Mat1-2, but there were no dual maters. Most conidia from dual maters also had one or the other of the mating-type genes, but not both. Thus, dual maters appear to lose one of the mating-type genes during vegetative growth. The incidence of self-mating in dual maters appears to depend on the co-occurrence of strains with each mating type in vegetative cultures. In rare transformants, the incoming sequences had replaced the resident mating-type gene. Nearly isogenic pairs produced from three M. grisea laboratory strains were mated to investigate their fertility. One transformant with switched mating type appears to have a mutation that impairs the development of asci when its mating partner has a similar genetic background. The M. grisea Mat1-1 and Mat1-2 genes are idiomorphs approximately 2.5 and 3.5 kb in length, respectively.