Intraspecific ITS polymorphism in Scutellospora castanea (Glomales, Zygomycota) is structured within multinucleate spores.

Polymorphism of the internal transcribed spacers (ITS) of the ribosomal DNA in Scutellospora castanea (Glomales, Zygomycota) and its organization among spores were evaluated. Polymerase chain reaction (PCR) amplification with ITS1/ITS4 primers yielded several fragments of different lengths, even from single spores. Fragments produced from multisporal DNA were cloned and grouped into 6 ITS types by PCR-RFLP and sequence analysis. Five type-specific primers were designed. Spores were then analyzed by PCR and amplification profiles revealed that they were qualitatively different one from another due to the presence or absence of some ITS types. Intrasporal segregation of ITS variant length types was also shown, by PCR experiments, utilizing diluted fractions of nuclear suspensions from single spores. The results demonstrate the mainly multikaryotic condition of the spores of S. castanea.

rDNA units are highly polymorphic in Scutellospora castanea (glomales, zygomycetes).

The ribosomal DNA (rDNA) units in the glomalean zygomycete fungus Scutellospora castanea were analyzed. Dot-blot assays allowed an estimation of 75 copies per genome. After constructing a genomic library in a phage lambdaEMBL3 vector, 13 rDNA clones were screened and explored. PCR experiments confirmed their nature and allowed homologous probes to be obtained. Restriction-fragment length polymorphism (RFLP) analysis and hybridizations with 18 s and 25 s probes allowed their grouping into nine families. The 18 s gene from these 13 clones was partially sequenced. The resulting 550 bases sequences were analyzed, and a phylogenetic tree was inferred. This revealed that two clones contain one highly divergent rDNA family (rUSc1) by comparison with other known 18 s sequences from the database. A phylogenetic tree was constructed with the entire 18 s sequences of rUSc1, rUSc3 and those of seven species representative of the glomalean fungi, Glomus, Entrophospora, Acaulospora, Scutellospora and Gigaspora. This tree confirmed that the rUSc1 sequence is the neighbor of 18 s sequences from Glomus (Glomineae), while rUSc3 remained in the group of the Gigaspora and Scutellospora (Gigasporineae). A specific primer, rUSc1-1, was generated from the ITS region of rUSc1, and used for PCR amplification from single spores, depicting the presence of rUSc1 in the genome of S. castanea at a lower frequency than other units.

Phylogenetic analysis of a dataset of fungal 5.8S rDNA sequences shows that highly divergent copies of internal transcribed spacers reported from Scutellospora castanea are of ascomycete origin.

Using a dataset comprising 5.8S rDNA sequences from a wide range of fungi, we show that some sequences reported recently from the arbuscular mycorrhizal (AM) fungus Scutellospora castanea most likely originate from Ascomycetes. Other ITS and 5.8S sequences which were previously reported are confirmed as being clearly of mycorrhizal origin and are variable within one isolate of S. castanea. However, these results mean that previous conclusions which were drawn regarding the heterokaryotic status of AM fungal spores remain unproven. We provide an enlarged 5.8S rDNA dataset that can be used to check ITS sequences for conflicts with well-established phylogenies of the organisms that they were obtained from.

A new species of Pythium isolated from the Burgundy region in France.

Pythium nodosum sp. nov. has been isolated from a soil sample taken in the Burgundy region in France. The fungus has spherical to variously shaped proliferating sporangia, smooth-walled oogonia which are crowded with different antheridial branches making a complicated knot around the former, and aplerotic oospores. Morphological and reproductive aspects of Pythium nodosum as well as the PCR of the internal transcribed spacer (ITS1) of the ribosomal nuclear DNA coupled with restriction fragment length polymorphism analysis are described here. The nucleotide sequences of ITS1 encoding 5.8S rRNA is also given.

Base composition of DNA from glomalean fungi: high amounts of methylated cytosine.

Glomales (Zygomycetes) are obligate fungal symbionts of roots of land plants and form arbuscular mycorrhiza. Sporal DNA of 10 isolates belonging to nine species was purified and the base composition was determined by RP-HPLC. Base composition fell in a narrow range between 30 and 35% G + C. A high amount of methylated cytosine (mC) accounting for 2-4% of the total nucleotides was found in all taxa. The DNA melting profile was defined for Scutellospora castanea. It corresponded to 32% G + C, and the shape of the denaturation curve suggested a heterogeneity in the GC content within the fungal genome. Knowledge of GC contents and variations between taxa are essential for evaluating nuclear DNA content using fluorimetric methods, and high proportions of mC/C + mC in the genomes of glomalean fungi could reflect the existence of repeated DNA families. Results are discussed in relation to data for other fungi and eukaryotes.

Characterization of a highly repeated DNA sequence (SC1) from the arbuscular mycorrhizal fungus Scutellospora castanea and its detection in planta.

A highly repeated DNA sequence from the genome of an arbuscular mycorrhizal fungus has been isolated and characterized. This 1,202-bp sequence (SC1) represents about 0.24% of the Scutellospora castanea genome, estimated to be 1 pg by flow cytometry. The sequence was shown to be a Scutellospora-specific probe in Southern blots and dot blot hybridizations. After complete sequencing of SC1, PCR primers were generated and used to amplify a 907-bp fragment from spores of S. castanea or from colonized Allium porrum roots. No amplification products were obtained with DNA from either spores or mycorrhizal root of other species of arbuscular mycorrhizal fungi. These primers were sufficiently specific for unequivocal detection of S. castanea in planta.

A point mutation in the gene encoding the Rubisco large subunit interferes with holoenzyme assembly.

Ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco), a key enzyme of photosynthetic CO2 fixation, is composed of 8 large and 8 small subunits. The Rubisco-deficient Nicotiana tabacum mutant Sp25 is able to synthesize the peptides for both subunits but does not contain any active holoenzyme. The phenotype is maternally inherited and thus caused by a mutation in the chloroplast genome, which also encodes the Rubisco large subunit. A comparison of the nucleotide sequences of the large subunit gene of the Sp25 mutant with that of the wild-type tobacco revealed a single nucleotide change in the Sp25 mutant. This resulted in an amino acid substitution at Gly-322, which was replaced by serine.

Origin, distribution and mapping of RAPD markers from wildPetunia species inPetunia hybrida Hort lines.

We have established the first linkage map forPetunia hybrida based upon both RAPD and phenotypical markers. The progeny studied consisted of 100 BC1 individuals derived from the [(St40xTlvl)xTlvl] back-cross. Each morphological marker has previously been mapped onto one of the seven chromosomes. The map consists of 35 RAPD loci of which 24 were affected onto chromosomes while 10 loci were not affected. The loci covered 262.9 cM with a mean distance of 8.2 cM. They are dispersed over seven linkage groups, of which six are carried on identified chromosomes. The RAPD markers were also applied on a set of tenP. hybrida, lines chosen for their diversity and on a set of seven wild species corresponding to the possible ancestors of theP. hybrida species. The markers were found both in the wild species as well as inP. hybrida lines indicating that they are inherited and are stable enough to establish similarities and to suggest relationships between species. Eight out of the ten lines carry different linkage groups of RAPD markers, which suggest that recombinant events occurred between chromosomes which originated in the wild species.

Genetic toxicity of methyl methanethiosulfonate on Salmonella typhimurium, Saccharomyces cerevisiae and Nicotiana tabacum.

The genetic toxicity of methyl methanethiosulfonate (MMT), a hydrolytic derivative of the insecticide methomyl, or lannate (Du Pont), was studied in Salmonella typhimurium, Saccharomyces cerevisiae and Nicotiana tabacum. At low concentrations, a lethal action was observed in all 3 models. Nuclear genetic effects of the product were not detected on the Xanthi x NC 95 hybrid of N. tabacum or on the diploid strain D7 of S. cerevisiae. At the cytoplasmic level, an effect on chloroplasts was observed on tobacco, but MMT did not induce cytoplasmic 'petites' in yeast. Ames tests with and without metabolic activations by S9 mix and/or fecalase were negative. MMT cannot be considered to be mutagenic on these models; consequently it is unlikely to be genotoxic in man, although these experiments do not exclude eventual co-genetic effects.

Biological study of tobacco seeds flown in the joint Apollo-Soyouz Test-Project.

A preliminary experiment was carried out in order to detect eventual effects attributable to primary and background cosmic radiations received by tobacco seeds during the joint Apollo-Soyouz space flight. No genetic effect was observed but several developmental and physiological alterations took place.