Molecular evolution of the fungi: relationship of the Basidiomycetes, Ascomycetes, and Chytridiomycetes.

Establishing the phylogeny of fungi and protists often has proved difficult owing to the simple morphologies and convergent characters in these organisms. We used DNA sequences of nuclear small-subunit ribosomal RNA genes to determine phylogenetic relationships among three major classes of organisms considered to be fungi--Basidiomycetes, Ascomycetes and Chytridiomycetes--and to assess the taxonomic position of Neocallimastix, an economically important anaerobic rumen microorganism whose classification is controversial. The Basidiomycetes and Ascomycetes, two classes of nonflagellated fungi, are the most closely related taxa. Chytridiomycetes, though bearing flagella, group with these higher fungi rather than with the protists. Neocallimastix, a eukaryote lacking mitochondria and variously classified as a protist or as a fungus, shows closest molecular affinities with the Chytridiomycete fungi in the order Spizellomycetales.     

西藏真菌增补

报道在西藏亚东、林芝等地区采集到的西藏新记录真菌34种。其中包括担子菌22种,子囊菌7种和粘菌5种。凭证标本存放在吉林农业大学真菌标本室(HJAU).     

A Cladistic Outline of the Eumycota

Abstract— A cladistic classification of fungi determined by a parsimony method with 21 terminal taxa and 51 characters is presented. Outgroup comparison with Oomycetes determined polarity assessments. The group Eumycota, including the traditional taxa Hyphochytriomycetes, Chytridio-mycetes, Zygomycetes, Ascomycetes and Basidiomycetes, is defined by two synapomorphies, molecular weight of 25S RNA, and chitin cell walls. Some groups are supported as monophyletic; Eumycota, Amastigomycota, Dicaryomycotina, Ascomycetes, Protobasidiomycetes, Basidiomycetes, Euascomy-cetidae, Hymenomycetidae and Homobasidiomycetales. The Hyphochytriomycota is the sister group to remaining groups. The Taphrinaceae and Saccharomycetaceae are more closely related to the Basidiomycetes than to any of the ascomycetous groups. In the absence of unique character sets groups such as the Mastigomycotina, Hemiascomycetes, Ustomycetes, Holobasidiomycetes, Heterobasidio-mycetes, Phragmobasidiomycetes and the Teliomycetes cannot be maintained and are abandoned as paraphyletic. Characters and terminal taxa used for the analysis are defined and discussed.     

Diversity and specific composition of microscopic fungi on synthetic polymeric materials

We summarized experimental data on species diversity of fungi decomposing synthetic polymeric materials. Most of the fungi were anamorphs of the phylum Ascomycota, class Ascomycetes (231 species and 85 genera). Teleomorphs of ascomycetes were represented by 18 species and 7 genera. We revealed a smaller number of fungi belonging to the phylum Zygomycota, class Zygomycetes (31 species and 15 genera), or the phylum Basidiomycota, class Basidiomycetes (5 species and 5 genera). The specific composition of fungi was assessed on polymeric materials of various classes.     

Preparative density gradient centrifugation of RNA and DNA in cesium sulfateurea mixture

A new procedure is described for a large scale separation and purification of unfixed DNA and RNA from a mixture of partially extracted nucleic acids and lysates of subcellular fractions by centrifugation to equilibrium in cesium sulfate-urea mixture. Optimum conditions are described for the separation and quantative recovery of both RNA and DNA in a pure form. The procedure allows determination of peak buoyant densities of 4–5s RNA, 7–11s mRNA and total cytoplasmic RNA. The procedure also allows fractionation of small molecular weight classes of cytoplasmic RNAs from the 18s and 28s rRNAs.     

Coprophilous fungi of the horse

A total of 1267 microfungi, including 35 Myxomycetes, were recorded from the fecal samples of the 60 horses; of these 395 were found on 20 saddle-horse feces, 363 on 20 race-horses and 509 on 20 working-horses. Eighty two species representing 53 genera were recorded; of these 7 were Zygomycetes, 18 Ascomycetes, 1 Basidiomycetes and 25 Fungi Imperfecti: 2 Myxomycetes. Common coprophilous fungi are in decreasing orderPilobolus kleinii, Saccobolus depauperatus, Mucor hiemalis, Lasiobolus ciliatus, Podospora curvula, Petriella guttulata, M. circinelloides, Coprinus radiatus, Dictyostelium mucoroides, Sordaria fimicola, C. miser, C. stercorarius, Acremonium sp., Coprotus granuliformis, Graphium putredinis, Iodophanus carneus, Chaetomium murorum, Podospora communis, P. inaequalis, P. setosa, Saccobolus versicolor andCladosporium cucumerinum. Species ofMyrothecium verrucaria, Actinomucor elegans, Kernia nitida, Spiculostilbella dendritica andMucorparvispora were found exclusively in working-horses feces.Badhamia sp., Anixiopsis stercoraria, Echinobotryum state ofD. stemonitis, Geotrichum candidum andOidiodendron sp. were found only in saddle-horses feces.Chlamidomyces palmarum andPhilocopra sp. were found exclusively in race-horses feces.Notes on infrequent or interesting fungi includeThamnostylum piriforme, Phialocephala dimorphospora, Rhopalomyces elegans andSpiculostilbella dendritica.     

MITOCHONDRIAL RNA FROM CULTURED ANIMAL CELLS : II. A Comparison of the High Molecular Weight RNA from Mouse and Hamster Cells

Discrete RNA fractions sedimenting slightly slower than 18s ribosomal RNA have been found in mitochondrial preparations from both hamster (BHK-21) and mouse (L-929) cells. This RNA could be separated into two components, present in approximately equimolar amounts, by prolonged zonal centrifugation or acrylamide gel electrophoresis. The hamster components had sedimentation constants averaging 16.8 and 13.4, and molecular weights (estimated by gel electrophoresis) averaging 0.74 and 0.42 x 10⁶ daltons. Mixed labeling experiments showed that the mouse components sedimented and electrophoresed 3–6% more slowly than the corresponding hamster components. The RNA from both cell lines resembled mitochondrial ribosomal RNA from yeast and Neurospora in being GC poor, and in addition the larger and smaller components resembled each other in base composition. These results, taken with those of other recent studies, are compatible with the idea that our high molecular weight mitochondrial RNA is ribosomal; such RNA would then constitute a uniquely small size-class of ribosomal RNA.     

Characterization of yeast ribosomal DNA fragments generated by EcoR1 restriction endonuclease

Summary The action of Escherichia coli restriction endonuclease R1 (EcoR1) on DNA isolated from Saccharomyces cerevisiae (strain MAR-33) generates three predominent homogenously sized DNA fragments (species of 1.8, 2.2 and 2.5 kilo nucleotide base pairs (KB). Many DNA species of molecular weight greater than 2 million daltons can be recognized upon incomplete EcoR1 digestion of yeast DNA. Four additional DNA species ranging from 0.3–0.9 KB can be identified as the second major class of EcoR1-yeast DNA products.Hybridization with radioactive ribosomal RNA (rRNA) and competition with nonradioactive rRNA show that of the three predominent EcoR1-yeast DNA species, the 2.5 KB species hybridizes only with the 25S rRNA while the lighter 1.8 KB species hybridizes with the 18S rRNA. The intermediate DNA species of 2.2 KB hybridizes to a small extent with the 25S rRNA and could be a result of the presence of the 2.5 KB DNA species. The mass proportions and hybridization values of these 3 DNA species account for about 60% of the total ribosomal DNA (rDNA).The 5EcoR1-yeast DNA species of less than 0.9 KB (4 major and 1 minor species) hybridize to varying degrees with the 2 rRNA and can be grouped in two classes. In one class there are 3 DNA species that hybridize exclusively with the 18S rRNA. In the second class there are 2 DNA species that besides hybridizing predominently with the 25S rRNA also hybridize with the 18S rRNA. The 7 EcoR1-yeast DNA species (excluding the 2.2 KB DNA species) that hybridize with the two rRNA account for nearly a 5 million dalton DNA segment, which is very close to the anticipated gene size of rRNA precursor molecule. If the 2.2 KB DNA species is a part of the rDNA that is not transcribed or 5 sRNA then the cistron encoding the rRNA in S. cerevisiae has at least 8 EcoR1 recognition sites resulting in 8 DNA fragments upon digestion with the EcoR1. Consideration is given to the relationship of the rRNA species generated by EcoR1 digestion and the chromosomes containing ribosomal cistrons.     

Deoxyribonucleic acid base composition and taxonomy in the genus Geotrichum Link

The molar percentage of guanine + cytosine (% GC) in the DNA from 25 yeast-like fungi related to the genus Geotrichum has been measured. This criterion together with the biochemical characteristics allow the division of the species into 3 groups: one group resembling G. candidum (% GC: 31.5–42) and containing 8 imperfect and 6 perfect forms. In this group only G. capitatum has a lower % GC (31.5). A second group of 6 species resembling the genus Trichosporon (57–60%) and a third intermediate category of 4 species (42–53.5%) were observed. The GC-values correlate well with biochemical characters, e.g. the presence of urease, the number of organic substrates assimilated, and also with the presence of characters typical of the Ascomycetes or the Basidiomycetes.This work was supported by I.N.S.E.R.M. (Institut National de la Santé et de la Recherche Médicale) C.R.L. n^o 76 1 153 8.     

Electrophoretic Analysis of Ribosomal and Viral Ribonucleic Acids with a Simple Technique for Slicing Low-Concentration Polyacrylamide Gels

The electrophoretic mobilities of ribosomal ribonucleic acids (RNA) from cultured mammalian (HeLa, Vero, MDBK), avian (chick embryo), and bacterial (Escherichia coli) cells, and RNA species extracted from selected viruses (Sindbis, polio, tobacco mosaic, Sendai) were compared, employing a simple, inexpensive technique for slicing low-concentration polyacrylamide gels. The procedure provides for rapid fractionation of gels used for characterization of RNA, incorporating extrusion and serial sectioning of frozen gels. Among 28S ribosomal RNA species, Vero and MDBK were indistinguishable, whereas HeLA RNA had a slightly lower mobility (higher apparent molecular weight) and chick RNA had a higher mobility (lower apparent molecular weight). The 18S ribosomal RNA species of the three mammalian sources were indistinguishable, but chick 18S RNA had a slightly lower apparent molecular weight. The inverse relation between mobility and log-molecular weight among the ribosomal and viral RNA species, though not highly precise, demonstrates the applicability of the technique to the study of molecular weights of viral RNA species.     

Isolation and characterization of cytoplasmic and chloroplastic ribosomes and their ribosomal RNAs from the diatom Cylindrotheca fusiformis

The cytoplasmic and chloroplast ribosomes from the marine diatom Cylindrotheca fusiformis were isolated and characterized. The cytoplasmic ribosomes sedimented in sucrose at 84S and dissociated into subunits of 64S and 42S in the absence of Mg²⁺. It contained ribosomal RNAs with molecular weights of 1.31×10⁶ and 0.70×10⁶. The chloroplast ribosomes sedimented at 70S only in the presence of high Mg²⁺ concentrations (25–100 mM). No stable subunits were routinely observed and at very high levels of Mg²⁺ (>100 mM) the 70S species was converted to a form sedimenting at 55S. At 4°C ribosomal RNAs with molecular weights of 1.1×10⁶ and 0.40×10⁶ were detected on polyacrylamide gel electrophoresis. When the RNAs were resolved at room temperature the large molecular weight component disappeared while RNA with molecular weights of 0.65×10⁶ and 0.53×10⁶ were observed. Apparently the large chloroplast RNAs dissociated into two pieces of unequal molecular weight. These properties of the diatom's chloroplast ribosomes are very similar to those of the counter parts in unicellular green algae, which suggests that both types of algae have a common phylogenetic ancestor.     

Genotypic analysis of variability in Zygomycetes. A mini-review

Different types of molecular markers are available for use in evolutionary and population studies of microscopic fungi. These approaches have proved their merits and have been successfully applied to a wide range of fungal species belonging in the Ascomycetes and Basidiomycetes. Species in the class Zygomycetes have been rather neglected from this aspect. This review discusses the information available from investigations of the genotypic variability in this group of fungi.     

Transfer RNAs as genotypic fingerprints of eubacteria

A new method was developed for rapid genotypic identification and classification of bacteria. The method is based on high resolution gel electrophoresis of the stable, low molecular weight (LMW) RNA fraction of single bacterial strains. This fraction comprises the total transfer RNA pool and the 5S ribosomal RNA. On a one-dimensional gel, every eubacterial strain exhibited a distinct LMW RNA profile, a set of bands belonging to three different size classes: 5S rRNAs (110–131 nt), class 2 tRNAs (82–96 nt) and class 1 tRNAs (72–79 nt). LMW RNA profiles of members of five of the ten major eubacterial groups, previously defined by 16S rRNA sequence analysis, were highly diverse. For some major groups, like flavobacteria and planctomyces, the distinctive sizes of their 5S rRNAs allowed the assignment of strains to these groups. More specific taxonomic information was gained from analysis of the tRNA part of the profile. Strains could be grouped as species and genera due to species- and genus-specific tRNA bands. From an evolutionary point of view, this order found in the total tRNA pool of eubacteria could indicate that cytoplasmic tRNA evolution reflects ribosomal RNA evolution. Given the universality of tRNAs, it is to be expected that their electrophoretic mobility profiles may serve as a convenient RNA fingerprint for defining bacterial species operationally and for identifying new genotypes by differing patterns.     

Kern-Plasma-Transport neusynthetisierter rRNS in Zellen einer Suspensionskultur von Petroselinum sativum

Summary A rapidly labelled rRNA precursor can be detected in callus cells of Petroselinum sativum grown on a liquid synthetic medium. Its molecular weight has been calculated to be 2.3×10⁶. This value agrees with that of the rRNA precursor from other plant material. In order to follow the synthesis and processing of rRNA in time and to correlate single steps in this process with cell organelles it was necessary to obtain pure fractions of nuclei and ribosomes. The isolation method for nuclei is given in detail. The nucleic acids are separated on polyacrylamide gels of low acrylamide concentration. Pulse-chase experiments show that the rRNA precursor is split into two fragments within the nucleus: an 18S and a 25S component. The 18S RNA leaves the nucleus rapidly. It is already found quantitatively in the ribosomal fraction after 30–60 min chase. At that time the 25S RNA is still within the nucleus; it appears much later in the ribosomes. Since the increase in ribosomal label occurs simultaneously with the decrease in nuclear label, it is concluded that there is no degradation of 18S RNA within the nucleus. Apparently there are two distinct transport mechanisms with different kinetics for the two RNA components.     

The relationship between ribosomal repeat length and genome size in Vicia

The organization of the ribosomal RNA genes was examined in several species of Vicia in an attempt to determine whether a relationship exists between genome size and ribosomal repeat length. Species within this genus exhibit a sevenfold variation in haploid DNA content. Our data suggest that species with an intermediate genome size maintain one predominant Eco RI class of ribosomal repeat of about 9 kilobases (kb). In contrast, the smallest and largest genomes of Vicia possess one major and several minor classes. The possible relationship between repeat classes among species is discussed. We examined the species with the smallest (V. villosa) and largest (V. faba) genomes in closer detail by R-loop analysis of a satellite DNA from Hoechst 33258 dye-CsCl gradients. Heterogeneity was found in the length of the ribosomal repeat for both species, but no appreciable difference was observed in the distribution of these lengths, which averaged 11–12 kb. This heterogeneity is associated with the nontranscribed spacer region. Intervening sequences were not found in either the 25S or 18S coding regions of the ribosomal repeat of either of these two plants. A putative ribosomal RNA precursor of 7 kb was identified for both species.     

The effect of reaction with formaldehyde on the sedimentation rates of ribonucleic acids

It has been reported that the RNA of several bacteriophages and that of the larger ribosomal sub-units of mammalian cells sediment faster in the presence of 0·1m-sodium chloride than is expected from their estimated molecular weights. The effect of blocking the hydrogen-bonding amino groups of these and other types of RNA was studied. The RNA of phage R17 no longer sedimented anomalously fast after treatment with formaldehyde. In contrast, the larger ribosomal RNA of HeLa cells appeared more aberrant than before, sedimenting faster than tobacco-mosaic-virus RNA (mol.wt. 2×10⁶) in the presence of formaldehyde. The rapidly labelled nuclear 45s RNA of HeLa cells still sedimented faster than the larger ribosomal RNA after reaction with formaldehyde, showing no evidence of disaggregation. It is suggested that both the large ribosomal RNA and the 45s RNA of HeLa cells may have a non-linear structure.     

On the number of ribosomal RNA Genes in man

Summary The saturation plateau of the high molecular weight species of ribosomal RNA was determined for DNA of human liver from a female and a male person with normal karyotypes. The amount of DNA annealing with (18s+28s) RNA of HeLa cells is equivalent to 443±21 or 416±19 copies of the corresponding gene loci, depending on the set of molecular weights of 18s and 28s RNA used in the calculation.

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Zusammenfassung Es wurden RNA-DNA-Hybridisierungsexperimente mit DNA aus der Leber einer Frau und eines Mannes mit normalem Karyotyp und dem Gemisch der beiden hochmolekularen Komponenten der ribosomalen RNA durchgeführt. Der Anteil der menschlichen DNA, der mit (18s+28s) RNA aus HeLa-Zellen hybridisiert, entspricht 443±21 bzw. 416±19 Kopien der für diese RNA-Species codierenden Gene, je nach der Größe der Molekulargewichte, die für die Berechnung verwendet werden.

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Supported by the Deutsche Forschungsgemeinschaft (SFB 46).     

Evolution of dark-spored Myxomycetes (slime-molds): molecules versus morphology

The Myxomycetes are a major component of soil amoebae, displaying a complex life cycle that terminates in the formation of often macroscopic fruiting bodies. The classification of Myxomycetes is controversial and strongly depends on the weight given by different authors to morphological and developmental characters. We used a molecular approach to establish the phylogenetic relationships in the dark-spored orders Stemonitales and Physarales. Twenty-five small subunit ribosomal RNA gene sequences were obtained, with focus on two Stemonitales genera, Lamproderma and Comatricha. Unexpectedly, our results show that Stemonitales are paraphyletic with Physarales arising from within a Lamproderma clade. The genus Lamproderma itself is polyphyletic and can be divided into two distinct clades. Additionally, we found that Comatricha nigricapillitia comprises two cryptic species, both related to Enerthenema. Our study allows the reappraisal of morphological and developmental characters in the light of molecular data and sets foundations for a new classification of Myxomycetes.     

5.3 S RNA is a discrete cleavage product from the 5′-terminus of 18 S RNA of rat liver ribosomes

A 5.3 S RNA species observed in urea-gel electrophoretic analysis of the RNA of the small ribosomal subunit of rat liver has been identified from its sequence as the 5′-terminal 133–134 base fragment of 18 S RNA. Presumably it is cleaved by an endogenous endonuclease when the ribosomal subunits are dissociated, because it usually is not observed in 18 S RNA obtained by direct extraction of cells or tissues.     

Purification and restriction endonuclease mapping of soybean 18 S and 25 S ribosomal RNA genes

The restriction endonuclease map of the 25 S and 18 S ribosomal RNA genes of a higher plant is presented. Soybean (Glycine max) rDNA was enriched by preparative buoyant density centrifugation in CsCl-actinomycin D gradients. The buoyant density of the rDNA was determined to be 1.6988 g cm^–3 by analytical centrifugation in CsCl. Saturation hybridization showed that 0.1% of the total DNA contains 25 S and 18 S rRNA coding sequences. This is equivalent to 800 rRNA genes per haploid genome (DNA content: 1.29 pg) or 3200 for the tetraploid genome. Restriction endonuclease mapping was performed with Bam H I, Hind III, Eco R I, and BstI. The repeating unit of the soybean ribosomal DNA has a molecular weight of 5.9·10⁶ or approximately 9,000 kb. The 25 S and 18 S rRNA coding sequences were localized within the restriction map of the repeating unit by specific hybridization with either [¹²⁵I]25 S or [¹²⁵I]18 S rRNA. It was demonstrated that there is no heterogeneity even in the spacer region of the soybean rDNA.