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PubMed1988

The effects of environmental stress on steroid receptor levels and steroid-induced morphogenesis in Achlya ambisexualis.

Incubation of Achlya ambisexualis at elevated temperature (heat shock) or in the presence of sodium arsenite resulted in an inhibition of steroid hormone-induced responsiveness. The effect of heat shock was time- and temperature-dependent and more severe than the effect of sodium arsenite. Incubation at 37 degrees C for 30 min completely abolished the steroid-induced response and full recovery was not observed until 6 h after a return to the normal growth temperature of 22 degrees C. Heat shock and arsenite treatment had no effect on the cellular uptake of the steroid hormone, but heat shock resulted in a time- and temperature-dependent loss in the cellular level of steroid receptors. In contrast, arsenite treatment had little effect on the concentration of steroid receptors. However, both heat shock and arsenite treatment produced a long-term (4 h) and transient (1 h) inhibition of total protein synthesis, respectively. The recovery of steroid-induced responsiveness following heat shock was observed after both protein synthesis and steroid hormone receptor levels had returned to normal values.

Fungi↗
PubMedDate not supplied

Synthesis of dehydro-oogoniol, a female-activating hormone of Achlya: the progesterone route.

The structure of dehydro-oogoniol (3 beta,11 alpha,15 beta,29-tetrahydroxystigmasta-5,24(28)(E)-dien-7-one), a female-activating hormone of the water mold Achlya, has been confirmed by synthesis. The starting material was progesterone, which was converted to the 11 alpha, 15 beta-dihydroxy derivative by microbiological hydroxylation with Aspergillus giganteus (ATCC 10059). The side chain was constructed in a stepwise manner by means of Wittig and Horner-Emmons reactions, and the C-7 ketone was then introduced by allylic oxidation. The biological activity of the synthetic compound was the same as that of the natural hormone.

Animals↗
PubMed1979

A freeze-fracture study of hormone-induced branching in the fungus Achlya.

The induction of the male sexual organ primordia (antheridial hyphae) by the steriod hormone antheridiol in the water mold Achlya ambisexualis Raper requires the production and secretion of the enzyme cellulase. It is postulated that a localized secretion of cellulase produces a limited area of wall hydrolysis that is blown out into a lateral bleb by turgor pressure. Freeze-etch preparations show membrane profiles similar to those seen in other systems where exocytosis is occurring. Such a mechanism would provide the required localized secretion of cellulase. Water stress, imposed by polyethylene glycol, prevents the formation of antheridial hyphae, the secretion of cellulase and the expected membrane profiles. After a period of recovery from water stress antheridial hyphae are formed, cellulase secretion occurs and the expected membrane profiles are restored.

Cellulase↗
PubMed1977

Relationship between nuclear and polysomal RNA populations of Achlya: a simple eucaryotic system.

The relationship between hnRNA and mRNA in the water mold Achlya has been investigated in several ways. Analysis of the nuclear and polysomal poly(A) RNA by sucrose density gradient centrifugation under denaturing and nondenaturing conditions showed that the populations have indistinguishable size distributions. The number average sizes were calculated to be 1150 nucleotides for nuclear and 1140 nucleotides for polysomal poly(A) RNA. Selective inhibition of rRNA synthesis was used to investigate the size distribution of hnRNA without regard to poly(A) content. Very little hnRNA was observed which sedimented more rapidly than polysomal poly(A) RNA. Hybridization experiments in which an excess of nuclear DNA was reacted with 3H-poly(A) hnRNA or 3H-poly(A) mRNA showed that both populations contain repetitive transcripts (9-10%) as well as single-copy transcripts (44%). Analysis of hybrids on hydroxyapatite in the presence of 8 M urea demonstrated that the poly(A) RNA complementary to repetitive DNA sequence components represented a population of molecules distinct from the population complementary to single-copy DNA. The complexity of whole cell, nuclear and polysomal RNA was determined by saturation hypbridization to single-copy 3H-DNA. All three populations were complementary to essentially the same fraction of the DNA. Terminal hybridization values were 3.84, 3.76 and 3.76% for whole cell, nuclear and polysomal RNA, respectively, representing a complexity of 2.1 X 10(6) nucleotides. These data suggest that the composition of the hnRNA and mRNA populations are essentially identical. No evidence for selective turnover of any sequence component or size class within the nucleus was observed.

Base Sequence↗
PubMed1981

Synthesis, glycosylation and rapid secretion of a glycoprotein by Achlya, a primitive eucaryote.

Achlya ambisexualis, a water mold, secretes several glycoproteins during exponential growth. Among these is a major protein of 39 000 daltons (protein A-39) which is secreted very rapidly. Protein A-39 is detected among the soluble cellular proteins labeled for 5 min. However, after longer labeling times, an additional 95 000 dalton glycoprotein was immunoprecipitated from among the cytoplasmic proteins by antiserum against protein A-39. This antiserum reacted with a single 37 000 dalton protein from the in vitro translation products of poly(A)-containing RNA in a wheat germ cell-free system which is cleaved to a faster moving component in the presence of dog pancreatic membranes. Immunoprecipitated, chain-completion products of polysomes also show a 37 000 dalton peptide which does not bind to lectins, indicating absence of co-translational cleavage and glycosylation. Tunicamycin inhibits the appearance of the 95 000 dalton protein. Several immunoprecipitable proteins, including protein A-39, having sizes identical to the secretory proteins accumulate in the cytoplasm in the presence of this inhibitor. A short pulse with [3H]glucosamine followed by a chase showed that incorporation in protein A-39 increases while that in 95 000 dalton protein is decreasing. These results suggest that the 95 000 dalton glycoprotein may serve as a glycosyl donor to secretory protein A-39.

Fungal Proteins↗
PubMed1987

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.

Chytridiomycota↗
PubMed1990

A method of sequencing without subcloning and its application to the identification of a novel ORF with a sequence suggestive of a transcriptional regulator in the water mold Achlya ambisexualis.

Genomic amplification with transcript sequencing (GAWTS) is a method of direct sequencing that involves amplification with PCR using primers containing phage promoters, transcription of the amplified product, and sequencing with reverse transcriptase. GAWTS requires the generation of PCR primers that are specific for the sequences on both sides of a region. Here we describe promoter ligation and transcript sequencing (PLATS), a direct method for rapidly obtaining novel sequences that utilizes generic primers and only requires knowledge of the sequence on one side of a region. PLATS involves restriction digestion of the amplified vector insert, ligation with a phage promoter, and then GAWTS using phage promoter sequences as the PCR primers. The method is rapid and economical because it uses a limited set of oligonucleotides, and it is potentially amenable to automation because it does not require in vivo manipulations. PLATS facilitates the determination of a genomic sequence responsible for cross-hybridization in a Southern blot. Using PLATS, sequence has been obtained from a 1.1-kb segment in Achlya ambisexualis, which cross-hybridizes to the DNA-binding region of the chicken and Xenopus estrogen receptors. To our knowledge, this represents the first sequence reported from the Oomycetes, a large and widely distributed group of fungi. The sequence reveals a large, transcribed open reading frame that is markedly deficient in the dinucleotide TpA. A putative zinc finger containing three cysteines and one histidine (C-X2-C-X12-H-X3-C) and an acidic segment hint that this clone may be a member of a novel class of transcriptional regulators.

Amino Acid Sequence↗
PubMed1978

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.

Base Sequence↗
PubMed1974

Sexual morphogenesis in Achlya: ultrastructural basis for the hormonal induction of antheridial hyphae.

Male strains of the water mold Achlya ambisexualis produce antheridial hyphae in response to the steroid hormone antheridiol. The antheridial hypha is postulated to be initiated through a localized wall softening with the enzyme cellulase. Freeze-etch studies of hormone-treated hyphae were conducted to determine if aggregates of vesicles are induced at the location of new antheridial hyphae. Localized aggregates of vesicles were found in conjunction with areas of wall thinning. These data suggest that the processes of vesiculation and secretion provide a mechanism for concentration of cellulase at the site of initiation of antheridial hyphae.

Cellulase↗
PubMed1983

Organization of Achlya mtDNA: a population with two orientations and a large inverted repeat containing the rRNA genes.

We have investigated mtDNA organization in the oömycetous water mold; Achlya, and report here that this primitive organism contains a circular mitochondrial genome of 49.8 kilobase pairs. Extensive restriction endonuclease analysis indicates that a significant portion of the genome is present as an inverted repeat. Of 52 restriction sites for 14 enzymes thus far mapped, 28 sites cluster in two 9.6-kilobase-pair regions; within these regions, the sequence of sites is inverted but the spacing between analogous sites is identical. The repeat arms have a maximum length of 12.1 kilobase pairs and are separated by 4.6-8.4 and 21.0-22.3 kilobase pairs of unique sequences. Transfer hybridization experiments show that genes for both the large and the small rRNAs are contained within each repeat. Restriction endonuclease analysis shows that the unique regions between the inverted repeats are present in both possible orientations with respect to each other and in approximately equal proportions. These orientational, or "flip-flop," isomers of the unique regions are postulated to occur by intramolecular homologous recombination between the repeated regions.

Base Sequence↗
PubMed1988

Transcellular proton current in Achlya bisexualis hyphae: relationship to polarized growth.

Growing hyphae of Achlya bisexualis drive an electric current through themselves, such that positive charge flows into the apical region (the anterior 300 micron) and exits distally along the hyphal trunk. They also generate a gradient of extracellular pH, such that the medium surrounding the apex is slightly alkaline whereas that along the hyphal trunk is acid. To explore the genesis of these gradients and their relationship to polarized extension, we examined the effects of changes in the composition of the growth medium. The transcellular electric current was most pronounced in medium rich in amino acids. In leaner medium, containing limited amounts of amino acids or none at all, the current was attenuated or absent. We interpret the results to mean that inward current represents H+/amino acid symport, mediated by porters that are preferentially localized in the apical region. Apical alkalinity may be due to ammonia production. Outward current, and perhaps also the generation of metabolic acid, reflects the distribution of the H+-ATPase, which is excluded from the apex but is abundant along the hyphal trunk. Thanks to the spatial segregation of transport functions, protons characteristically flow into the apical region. However, since hyphae grow apically and at the same rate despite wide variations in current pattern, the flow of electric charge through the hyphae cannot be required to polarize extension or to localize the tip.

Amino Acids↗
PubMed1992

Extension growth of the water mold Achlya: interplay of turgor and wall strength.

When hyphae of the water mold Achlya were subjected to osmotic stress, imposed with polyethylene glycol (PEG)-300 or sucrose, turgor pressure fell in proportion to the increase in external osmotic pressure. There was no evidence of turgor regulation, even over a period of days, yet the extension rate was unaffected until turgor was reduced to less than a third of the normal level of 0.6-0.8 MPa (6-8 bars). Measurements of the pressure at which the hyphae burst indicate that they respond to osmotic stress by softening their apical cell walls, sustaining extension growth despite reduced turgor pressure. The effect of osmolytes excluded by the wall was very different; superfusion of growing hyphae with PEG-6000 or dextran-6000 reduced turgor and stopped extension but did not induce wall softening. Furthermore, the hyphae did not resume growth during an hour or more of continuous exposure to these substances. Although the two classes of osmolytes have the same effect on turgor, they may induce different strains within the cell wall; this might then affect the capacity of the organism to detect the drop in turgor or to soften its cell wall. The interplay between turgor and wall strength supports the proposition that turgor supplies the driving force for extension and that production of the standard hyphal form requires a balance between hydrostatic pressure and a resistive cell wall.

Journal Article↗
PubMed2002

A. robusta sp. nov., a new species of Achlya (Saprolegniales, Straminipila) from a polluted Argentine channel.

Achlya robusta sp. nov. was found on litter (floating twigs, leaves, and roots) in an artificial polluted channel, near a petroleum refinery, in Buenos Aires province, Argentina. The species is described, illustrated and compared with other species of the genus. A. robusta produces mainly smooth and papillate, tuberculate or bullate oogonia and monoclinous antheridial branches. It develops spherical and subglobose oogonia, with the oogonial wall yellowish and containing mainly immature oospheres. The oospores are eccentric and ranging from (1) 4-17 (30) per oogonium.

Argentina↗
PubMed1984

Transcellular ion currents in the water mold Achlya. Amino acid proton symport as a mechanism of current entry.

Achlya, like other tip-growing organisms, generates an endogenous electrical current such that positive charge flows into the hyphal apex and exits from the trunk. The present study is concerned with the mechanism of current generation by hyphae growing in a defined, complete medium. The intensity of the current, measured in the extracellular medium with a vibrating probe, was unaffected by the removal of all the inorganic constituents of the growth medium. However, an increase in the external pH or the deletion of amino acids abolished the current. Removal of methionine alone diminished the current by two thirds. Hyphae also generated a longitudinal pH gradient in the extracellular medium; the region surrounding the tip was more alkaline than the bulk medium, whereas the region around the trunk was relatively acidic. These findings suggest that a flux of protons, dependent upon amino acids in the medium, carries current into the tip and creates the surrounding alkaline zone. The proton current appears to result from the transport of amino acids rather than their metabolism. Conditions that abolished the current also inhibited methionine uptake but had little effect on the respiratory rate. The findings imply a connection between the proton current and chemiosmotic energy transduction. We propose that protons flow into the hyphal tip through amino acid/proton symporters that are preferentially localized there. The proton flux energizes the uptake of amino acids into the growing zone and may also contribute to the polarization of hyphal growth.

Amino Acids↗
PubMed1986

Ionophores and cytochalasins modulate branching in Achlya bisexualis.

Hyphae of Achlya bisexualis growing on a medium deficient in amino acids elongated but produced relatively few branches. Branching was enhanced by three classes of compound: cytochalasins A and E, the calcium ionophores A23187 and ionomycin and proton ionophores such as tetrachlorosalicylanilide (TCS), carbonylcyanide m-chlorophenylhydrazone (CCCP), and carbonylcyanide p-trifluoromethoxyphenylhydrazone (FCCP). We suggest that the effects of cytochalasins reflect the disruption of a microfilament-based system for vesicle transport. Enhancement of branching by ionophores implicates cytoplasmic ions in the control of branch initiation. There may be links between these phenomena and the earlier discovery that a new point of proton entry precedes the emergence of a branch and predicts its locus.

Calcimycin↗
PubMed1988

Transcellular ion currents during sporangium development in the water mould Achlya bisexualis.

Changes in the pattern of electric currents that accompany the transformation of growing hyphae of Achlya bisexualis into sporangia have been examined. When hyphae were transferred to a non-nutrient buffer, they continued to extend for several hours and then gave rise to sporangia. Throughout this process, current (positive charge) flowed into the apical region that corresponds approximately to the future sporangium. The current ceased after the crosswall appeared. The sporangium then remained electrically quiescent, except for a brief intense burst of outward current at the 'homogeneous' stage of spore cleavage. The inward current during sporangium formation largely represents an influx of protons. Addition of nitrate abolished the flow of electric current with little effect on sporulation. The late burst of outward current is most probably an artefact, generated by the discharge of salts from the sporangial vacuole. The transcellular electric current apparently plays no role in sporangium formation or in spore cleavage. Calcium ions, however, are required and may traverse the plasma membrane.

Calcium↗
PubMed1988

DNA-mediated transformation in the aquatic filamentous fungus Achlya ambisexualis.

A DNA-mediated transformation system was developed for the aquatic filamentous fungus Achlya ambisexualis using the chimeric plasmid vector pSV2neo2 microns. Hyphal colonies resistant to the neomycin analogue G-418 sulphate were regenerated from transformed protoplasts on soft agar. Southern blot analyses of the transformed-cell DNA produced multiple hybridization bands, suggesting integration of vector DNA into the host genome at multiple sites. Northern blot analyses revealed the presence of three APHII-gene-specific transcripts in the transformant, indicating that the G-418-resistant phenotype was due to the expression of the APHII gene. The presence of multiple RNA transcripts of unexpectedly large size suggested that RNA initiation and/or termination is under the control of regulatory element(s) other than the SV40 promoter. Plasmid DNAs recovered by transformation of Escherichia coli cells with total DNA preparations from the fungal transformants showed considerable DNA rearrangements. However, at least a portion of the plasmid DNA recovered from each of the transformants carried a functional APHII gene, suggesting that the episomal vector DNA may have played a role in maintaining the G-418-resistant phenotype.

Chytridiomycota↗
PubMed1989

Chemotropism and branching as alternative responses of Achlya bisexualis to amino acids.

Hyphae of Achlya bisexualis growing on lean media orient their extension towards a source of amino acids, and also put forth branches. Micropipettes were used to generate gradients of amino acids in the vicinity of individual hyphae. Phenylalanine and methionine were the most powerful attractants: 0.04 mM amino acid in the pipette produced reorientation, and higher concentrations made the hyphae curl around the pipette and grow into its tip. Hyphae detected gradients as low as 5% across their width. Methionine and phenylalanine appeared to bind to different receptors. Local application of these amino acids also elicited the emergence of single branches, next to the pipette and on the high side of the gradient; comparison of diverse amino acids and their analogues suggested that branching and chemotropism share common receptors. By contrast, cytochalasin A and various ionophores induced branches at random sites, without receptor involvement. We propose that binding of amino acids to their receptors determines the site of precursor vesicle exocytosis, and consider possible mechanisms.

Amino Acids↗