Julie C. Silver

DNA restriction fragment length polymorphisms in the rDNA repeat unit of Entomophaga

A heterologous rDNA probe was used to detect restriction fragment length polymorphisms in Entomophaga rDNA sequences. Six Canadian strains of Entomophaga aulicae, isolated from the spruce bud worm or hemlock looper in Ontario or Newfoundland, showed no detectable rDNA variation at 10 different restriction enzyme loci: BamHI, DraI, EcoRI, EcoRV, HindIII, HinfI, MspI, PstI, RsaI, or TaqI. A total of 14 isolates of E. aulicae representative of several different geographic and host origins were compared at the DraI and HindIII rDNA loci and two different banding patterns were detected. Of these, 12 showed the same patterns and were designated E. aulicae type 1. The two members which differed were designated E. aulicae type 2. The variations in rDNA restriction sites did not appear to be geographically dependent. Entomophaga maimaiga, a recently reclassified species from the E. aulicae complex, displayed an rDNA banding pattern clearly distinguishable from the E. aulicae patterns with DraI, EcoRI, EcoRV, or HindIII. Members of the E. grylli species complex exhibited patterns which clearly differed from the patterns seen with either E. aulicae or E. maimaiga isolates. However, members of the E. grylli species complex appeared to be more heterogeneous than those in the E. aulicae complex. Among four E. grylli members, three different rDNA banding patterns were detected with either HindIII or DraI. These were designated as E. grylli type 1, type 2, and type 3. An undesignated Entomophaga isolate from a dipteran host displayed rDNA polymorphisms not previously noted in either the lepidopteran or orthopteran isolates. Our results suggest that RFLPs in rDNA are useful in the delineation of genera and species within the Entomophthorales, but may not be as useful at lower taxonomic levels. These and other RFLPs can however provide useful information regarding the epidemiology of Entomophaga epizootics.

Cellular localization of steroid hormone-regulated proteins during sexual development in achlya

In the fungus Achlya ambisexualis sexual development in the male strain E87 is controlled by the steroid hormone antheridiol. To investigate the effects of antheridiol on the synthesis and/or accumulation of specific cellular proteins we have analysed [35S]methionine-labeled proteins from control and hormone-treated cells using both one-dimensional (1D) and two-dimensional (2D) PAGE. Since in a total cell extract, hormone-induced changes in specific proteins might not be apparent against a background of more abundant proteins, cells were fractionated prior to protein isolation. It was also necessary to establish a concentration of hormone carrier, in this case methanol, which by itself did not alter the pattern of protein synthesis. Using these approaches the addition of the hormone antheridiol to vegetatively growing cells of Achlya E87 was found to result in changes in the synthesis and/or accumulation of at least 16 specific proteins, which could be localized to the cytoplasmic, nuclear or cell wall/cell membrane fractions. The most prominent changes observed in the hormone-treated cells included the appearance in the cytoplasmic fraction of labeled proteins at 28.4 and 24.3 kD which were not detectable in control cells, and a significant enrichment in the labeling of a 24.3 kD protein in the cell wall/cell membrane fraction. A marked increase in the labeling of 85, 63 and 47 kD proteins in the nuclear fraction from hormone-treated cells was also noted. The molecular weight (MW) and the behavior on 2D gels of the 85 kD hormone-induced protein appeared very similar to that of the 85 kD heat-shock protein reported in Achlya. Quantitive changes in the [35S]methionine labeling of several other proteins were noted in all three cell fractions.

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 Oömycete 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.

Molecular cloning and characterization of two distinct hsp 85 sequences from the steroid responsive fungus Achlya ambisexualis

SummaryIn Achlya ambisexualis, hsp85 is one of the characteristic mycelial heat shock proteins induced in response to a rapid elevation in temperature (Silver et al. 1983). This heat shock protein has the same electrophoretic mobility on two-dimensional gels and is antigenically related to an 85 kDa steroid hormone-regulated protein which constites a component of the putative Achlya steroid hormone-receptor complex. We report here the isolation of two distinct, yet highly related, hsp85 gene sequences from Achlya genomic libraries. Northern analyses, using these two Achlya genomic sequences as probes, suggest that there are two hsp85 message population in Achlya and that at least one of these is regulated by the steroid hormone antheridiol.

Sympatric occurrence of two Entomophaga aulicae (Zygomycetes: Entomophthorales) complex species attacking forest lepidoptera

Abstract Entomophthoralean fungi caused infections in sympatric larval populations of Lymantria dispar and Heterocampa guttivitta in New York and Vermont during 1989 and 1990. Gross morphology as well as restriction fragment length polymorphism and allozyme analyses determined that the Japanese fungus Entomophaga maimaiga was responsible for L. dispar mortality, while a different and otherwise unidentified member of the E. aulicae species complex was responsible for H. guttivitta mortality. Bioassays confirmed that the fungus infecting H. guttivitta was not E. maimaiga; the fungal isolate from H. guttivitta could not infect L. dispar larvae.

Analysis of histones associated with neuronal and glial nuclei exhibiting divergent DNA repeat lengths.

Abstract: Total cerebral hemisphere nuclei purified from adult rabbit brain were subfractionated into neuronal and glial populations. Previous studies have shown that chromatin in neuronal nuclei is organized in an unusual nucleosome conformation compared with glial or kidney nuclei, i.e., a short DNA repeat length is present. We now analyze whether this difference in chromatin organization is associated with an alteration in the histone component of nucleosomes. Total histone isolated by acid/urea‐protamine extraction of purified neuronal, glial, and kidney nuclei was analyzed by electrophoresis on SDS‐polyacrylamide slab gels. Histone H1 that was selectively extracted from nuclei was also examined. Differences were not observed on SDS gels in the electrophoretic mobilities of histones associated with either the nucleosome core particle (histones H2A, H2B, H3, H4) or the nucleosome linker region (histone H1). Total histone and selectively extracted histone H1 were also analyzed on acid/urea slab gels that resolve histones on the basis of both molecular weight and charge differences. When analyzed in this system, differences with respect to electrophoretic mobility were not detected when comparing either selectively extracted histone H1 or total histone from neuronal and glial nuclei. Quantitative analyses were also performed and neuronal nuclei were found to contain less histone H1 per milligram DNA compared with glial or kidney nuclei. Neuronal nuclei also demonstrated a lower ratio of histone H1/core histone. These results suggest that the pronounced difference in chromatin organization in neuronal compared with glial nuclei, which is reflected by a short DNA repeat length in neurons, appears to be associated with quantitative differences in neuronal histone H1.

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.

Steroid hormone-regulated basic proteins inAchlya ambisexualis

We have investigated specific basic proteins in the oomyceteAchlya ambisexualis, strain E87, which are synthesized and/or accumulate in response to the steroid hormone antheridiol. Three hormone-regulated basic proteins were identified. One of the three basic proteins (64 kDa) was seen only in the cytoplasmic fraction, another (36 kDa) appeared unique to the cell wall/cell membrane fraction, while a third basic protein (63 kDa) was found in both cell fractions. Taken together with previous studies, these results indicate that antheridiol regulates a group of at least four distinct proteins in the molecular weight range of 63,000 to 64,000 which differ in their isoelectric points and in their cellular localization.

Hsp90-containing multiprotein complexes in the eukaryotic microbe Achlya

In the oomycete fungus Achlya ambisexualis, hyphae of the male strain undergo sexual differentiation in the presence of the steroid hormone antheridiol. Earlier studies demonstrated that antheridiol binds with high affinity to a 9S multiprotein complex from A. ambisexualis cytosols. Although these complexes were found to contain the heat shock protein Hsp90, the other components were not known. It was of interest to determine if any of the other protein components in the Achlya Hsp90-heterocomplexes would be homologous to those found in the steroid receptor-Hsp90-heterocomplexes of vertebrates. Cytosolic proteins of 110 kDa, 74 kDa, 64 kDa, 61 kDa, 56 kDa, 47 kDa, 27 kDa and 23 kDa, were found in repeated trials, to co-immunoprecipitate with Achlya Hsp90. The 74 kDa protein was identified as the heat shock protein Hsp70, the 23 kDa protein was found to be related to the vertebrate protein p23 and the 56 kDa protein was found to be related to immunophilin FKBP51. All three of these proteins are components of the vertebrate receptor heterocomplexes. The 110 kDa, 61 kDa and 27 kDa proteins appeared to be unique to the Achlya complexes. Unlike the seven other proteins co-immunoprecipitating with Hsp90, the 61 kDa protein was observed only in the co-immunoprecipitates produced from in vitro translates of RNA isolated from antheridiol-treated mycelia.