Expression of herpes simplex virus thymidine kinase gene in aquatic filamentous fungus Achlya ambisexualis

We have constructed a plasmid vector pSV2neo-MK alpha G in which the structural tk gene for Herpes simplex virus thymidine kinase (HSV-TK) was placed downstream from the metallothionein-I promoter. The vector also contained the selection marker aminoglycoside 3'-phosphotransferase (Km). This vector was able to transform the filamentous fungus Achlya ambisexualis and G-418-resistant colonies were obtained. Southern blot analyses revealed that multiple bands hybridizing to the HSV tk gene probe were present in the genomic DNA of the transformants. Upon analysis by gel electrophoresis, one of the transformants exhibited TK activity bearing electrophoretic mobility similar to that of the HSV-TK. An increase of approx. 40% of [3H]thymidine uptake and incorporation into cellular DNA was also observed in this transformant. This study suggested that the HSV tk gene can be expressed in the fungus A. ambisexualis that can be considered as a candidate host cell for further gene-expression studies.     

Basic nuclear proteins in the aquatic fungus Achlya ambisexualis.

The complement of basic chromosomal proteins in the aquatic fungus Achlya ambisexualis has been characterized. Achlya nuclei contain proteins with electrophoretic mobilities on acetic acid/urea and dodecyl sulphate polyacrylamide gels which are comparable to rabbit kidney histones H3, H4 and H2A. In contrast, the behavior of putative H2B and H1 proteins from Achlya showed greater analogy on acid/urea gels to higher plant histones. A closely related water fungus Saprolegnia ferax contained basic nuclear proteins which were very similar to those of Achlya.     

Purification and characterization of RNA polymerase II from the aquatic fungus,Achlya ambisexualis

The DNA-dependent RNA polymerase II or B (ribonucleotide-triphosphate: RNA nucleotidyl transferase, EC 2.7.7.6) from the Oomycete fungusAchlya ambisexualis has been purified to apparent homogeneity. The purification procedures involve precipitation with polyethylenimine, selective elution of RNA polymerase from the polyethyleneimine precipitate, ammonium sulfate fractionation, DEAE-cellulose chromatography, CM-cellulose chromatography, and chromatography on DNA-Sepharose 4B affinity columns. Utilizing these procedures 3 mg of RNA polymerase II is recovered from 1.6 kg of mycelium (wet weight). Purified RNA polymerase II fromA. ambisexualis was half-maximally inhibited by the mushroom toxin α-amanitin at a concentration of 0.046 μg/ml (5 × 10⁻⁸m). A second RNA polymerase activity is half-maximally inhibited at 55.6 μg/ml (6 × 10⁻⁵m). RNA polymerase II fromAchlya has 13 subunits with the following molecular weights: 180,000; 140,000; 99,000; 89,000; 69,000; 53,000; 41,000; 35,000; 29,000; 25,000; 19,000; 16,500; and 14,000. With regard to template preference, salt optima, and divalent metal cation optima,Achlya RNA polymerase is quite typical of other eucaryotic RNA polymerases.     

Hormone-dependent lipoxygenase activity in Achlya ambisexualis

Homogenates of male Achyla ambisexualis oxidize exogenously added [¹⁴C]arachidonic acid to an unidentified lipoxygenase product. Synthesis occurs at a rate of 10.6 ± 1.3 μg mg⁻¹ protein 30 min⁻¹. Activity in homogenates of female mycelium is only 2.1 ± 1.2. Conversion is eliminated by the lipoxygenase inhibitor nordihydroguaiaretic acid (10⁻⁴ M). Homogenates prepared from the male grown in chemical but not physical contact with female mycelium had decreased lipoxygenase activity (3.1 ± 1.5), suggesting that antheridiol produced by the female decreases lipoxygenase activity in the male. To confirm this, actively growing male cultures were exposed to 10⁻⁹ M 7-deoxy-7-dihydroantheridiol, a stable analog of antheridiol, for 24 h. Homogenates from these cultures also had diminished lipoxygenase activity (2.7 ± 1.0). 7-Deoxy-7-dihydroantheridiol added to the incubation mixture at 10⁻⁹ M had no effect on lipoxygenase activity (9.0 ± 1.8), excluding a direct action of 7-deoxy-7-dihydroantheridiol on the enzyme. These data support the hypothesis that lipoxygenase products are associated with vegetative growth and suggest the antheridiol initiation of reproductive growth suppresses lipoxygenase activity.     

Steroid hormone-induced changes in secreted proteins in the filamentous fungus Achlya

In the fungus Achlya, sexual development in the male strain E87 is mediated by the steroid hormone antheridiol. Treatment of vegetatively growing cells of E87 with antheridiol resulted in changes in the [35S]methionine labeling of several secreted proteins. The most heavily labeled group of proteins observed in the secreted fraction from control cells appeared on one-dimensional gels as a prominent wide band which could be resolved into three closely spaced components with relative molecular weights (MWs) of 57, 54, and 50 kD, respectively. After hormone treatment the two lower MW proteins of the group were barely detectable. Concomitant with the observed reductions in the labeling of the 54 and 50 kD proteins was the increased labeling of a doublet of very prominent proteins with relative MWs of 44.4 and 43 kD, respectively. The results of experiments with Endoglycosidase H suggested that the 44.4 and 43 kD proteins seen in hormone-treated cultures, might result from the removal or reduced synthesis of high mannose oligosaccharide groups found on the 54 and 50 kD proteins normally seen in control cultures. Additional support for this suggestion was provided by the observation that the 54 and 50 kD proteins from control cultures appeared to bind to conA columns and to be eluted with alpha-methylmannoside, while there was little or no binding of the 44.4 and 43 kD proteins from hormone-treated cells. Although other possibilities are not excluded, the results are suggestive of a steroid hormone-induced alteration in glycoprotein processing. The functions of the observed hormonally-responsive secreted proteins as well as those of the secreted proteins in non-hormone-treated cultures, are not known at this time.     

Chromatin organization in the oomycete Achlya ambisexualis.

Nuclei from the O?mycete Achlya ambisexualis and rabbit kidney nuclei were digested with micrococcal nuclease and the resultant DNA fragments analyzed on slab gels. The average DNA repeat size was found to be 159 +/- 1.2 base pairs for Achlya and 199.8 +/- 3.7 base pairs for rabbit kidney. The presence of a DNA repeat size of 159 base pairs for Achlya extends the characterization of eukaryotic chromatins to this most primitive and perhaps unique microbe.     

Analysis of the steroid receptor of Achlya ambisexualis

We have previously reported the discovery of a specific high-affinity binding protein for the fungal sex steroid pheromone antheridiol in the cytosol of Achlya ambisexualis male cells. In this report, we describe the fractionation of the binding protein from the cytosol by ammonium sulfate precipitation, the optimization of in vitro conditions for radioligand binding assays, and some of the biochemical properties of the binding protein. In the presence of sodium molybdate, the macromolecule has a sedimentation coefficient of 8.3 S in sucrose gradients of low ionic strength, a Stokes radius of 56.6 A (Sephacryl S-300 columns), a molecular weight of approximately 192,000, a frictional ratio of 1.5, and an axial ratio of 8.9. The binding protein can be eluted with 0.24 M KCl as a single peak from DEAE-Sephadex A-25 columns. These results indicate that this steroid-binding protein from a primitive eukaryotic microbe has in vitro biochemical properties that are similar to those of other known steroid receptors in higher organisms.     

Cellulase Localization in Hyphae of Achlya ambisexualis

Cellulase (EC 3.2.1.4; beta-1, 4-glucan glucanohydrolase) was localized at the ultrastructural level and found to occur in dictyosomes and vesicles, around the periphery of unidentified storage bodies, between the plasmalemma and the cell wall, and on the outer surface of the cell wall in the male strain (E87) of Achlya ambisexualis after treatment with the sex hormone antheridiol.     

Diversity and abundance of polyadenylated RNA from Achlya ambisexualis

The diversity, abundance, and DNA sequence representation of poly(adenylic acid) containing RNA derived from cells of Achlya ambisexualis cultured in defined and undefined media have been determined. The kinetics of hybridization of polyadenylated RNA with complementary DNA were the same for both culture conditions and revealed the presence of three frequency classes containing 29, 220, and 3000 different sequences of an average length of 1150 nucleotides. Complexity estimates derived from experiments in which polyadenylated RNA was hybridized to unique sequence DNA were in good agreement with these results. The kinetics of hybridization of complementary DNA with an excess of nuclear DNA indicate that approximately 10% of the RNA is transcribed from reiterated DNA sequences while the remainder is transcribed from single copy sequences.     

Hormonal stimulation of ribosomal RNA synthesis in Achlya ambisexualis.

The experiments reported show that one of the early effects of the steroid sex hormone antheridiol is on the synthesis of rRNA and ribosomes. This is demonstrated in hormone treated cultures of Achlya ambisexualis (strain E87) by an enhancement of the incorporation of [3H]uridine into 26S and 18S rRNA and by an increase in measurable amounts of ribosomes per milligram dry weight of mycelium. Furthermore, since the hormone does not significantly alter the pool size or the specific activity of uridine triphosphate, this effect appears to represent an increased rate of RNA synthesis.     

Cellulase Induction and Wall Extension in the Water Mold Achlya ambisexualis

Branching of the coenocytic hyphae in the water mold Achlya ambisexualis Raper is always accompanied by a rise in cellulase activity. Branching and the cellulase response can be induced either by the sexual hormone atheridiol or by casein hydro-lysate. Only those strains which branch in response to the inducer show a concomitant rise in cellulase. The peak of induced mycelial cellulase corresponds in time with the appearance of branch primordia.     

Synaptonemal complexes in antheridia of Achlya ambisexualis E87

This is the first report of longitudinal sections of synaptonemal complexes in oömycetous fungi. These indicators of meiosis were observed in antheridial nuclei of Achlya ambisexualis E87. They were attached to a platelike structure at the inner membrane of the nuclear envelope. The lateral elements were separated from each other by an average distance of 160 nm. These results provide new ultrastructural evidence for gametangial meiosis in Oömycetes.     

Microfilament bundles in antheridial nuclei of Achlya ambisexualis E 87

This is the first report of intranuclear microfilaments within gametangial nuclei of oömycetous fungi. Longitudinal sections of four to six microfilaments were frequently observed in meiotic antheridial nuclei of Achlya ambisexualis. Each microfilament measured approximately 7–10 nm in diameter. Spindle tubules (25 nm in diameter) were also observed within some of the nuclei possessing microfilaments.     

Agrobacterium-mediated transformation of the filamentous fungus Aspergillus awamori

Many transformation methods have been developed to introduce DNA into filamentous fungi. One of these methods is Agrobacterium-mediated transformation (AMT). Here, we describe an efficient protocol for AMT of Aspergillus awamori. This protocol has been used to determine the function of Agrobacterium virulence genes during AMT, to identify factors influencing transformation frequencies, to generate insertional mutants and to generate A. awamori gene knockout transformants. This protocol in not only applicable to A. awamori, but can be used as a more general guideline for AMT of other filamentous fungi. Conidiospores are incubated with induced Agrobacterium, and, after a cocultivation and selection period, hygromycin-resistant transformants are obtained with a frequency of 200-250 transformants per 1 x 10(6) conidiospores. Using this protocol, transformants can be obtained within 10-12 d.     

Calmodulin from the water mold Achlya ambisexualis: isolation and characterization

A protein-activator of bovine cyclic nucleotide phosphodiesterase from the water mold Achlya ambisexualis has been affinity-purified to apparent electrophoretic homogeneity. The heat-stable protein is similar in amino acid content and electrophoretic mobility on SDS acrylamide gels, to bovine brain calmodulin. It also cross-reacts with antibodies raised to the bovine protein. Achlya calmodulin activates PDE increasing its activity up to 9-fold in a Ca2+-dependent manner. The mold protein appears unusual in that its tyrosine fluorescence is unaltered by Ca2+ or by EGTA.     

Effect of culture medium composition on pheromone receptor levels in Achlya ambisexualis

Sexual reproduction in the eukaryotic fungi Achlya is controlled by two steroid pheromones. Antheridiol is the steroid released by female cells that induces male sexual differentiation. The antheridiol-induced response of male cells has been shown to be influenced by the composition of the culture medium. The present study was designed to determine if the composition of the culture media might also affect the levels of antheridiol binding protein in the cytosol of male cells. The mycelial content of cytosolic steroid pheromone binding sites in Achlya ambisexualis E87 males was measured at daily intervals during 6 days of suspension culture in media containing different nitrogen sources. Levels of binding sits increased during the first 2 days in culture to a plateau that was maintained for the next 2-3 days. During the first 3 days in culture, levels were much lower in mycelia cultured in an enriched medium containing lactalbumin hydrolysate compared to mycelia cultured in defined media containing glutamic acid as the nitrogen source. The level of binding sites increased rapidly when mycelia were transferred from an enriched medium to a nutrient-free salt solution and decreased when mycelia were transferred from a defined to an enriched medium. The relative differences in cytosolic binding measured by in vitro radioligand saturation analysis were confirmed by in vivo uptake studies. It is concluded that the mycelial content of antheridiol binding sites can be experimentally manipulated by variations in the composition of the culture medium and/or the time period of incubation in the medium.     

Agrobacterium tumefasciens-mediated transformation of the aquatic fungus Blastocladiella emersonii

Agrobacterium tumefaciens is widely used for plant DNA transformation and more recently, has also been used to transform yeast, filamentous fungi and even human cells. Using this technique, we developed the first transformation protocol for the saprobic aquatic fungus Blastocladiella emersonii, a Blastocladiomycete localized at the base of fungal phylogenetic tree, which has been shown as a promising and interesting model of study of cellular function and differentiation. We constructed binary T-DNA vectors containing hygromycin phosphotransferase (hph) or enhanced green fluorescent protein (egfp) genes, under the control of Aspergillus nidulans trpC promoter and terminator sequences. 24 h of co-cultivation in induction medium (IM) agar plates, followed by transfer to PYG-agar plates containing cefotaxim to kill Agrobacterium tumefsciens and hygromycin to select transformants, resulted in growth and sporulation of resistant transformants. Genomic DNA from the pool o resistant zoospores were shown to contain T-DNA insertion as evidenced by PCR amplification of hph gene. Using a similar protocol we could also evidence the expression of enhanced green fluorescent protein (EGFP) in zoospores derived from transformed cells. This protocol can also open new perspectives for other non-transformable closely related fungi, like the Chytridiomycete class.