Cytofluorometric determination of relative DNA content in nuclei of Coniophoraceae (Boletales) using DAPI

A new method based on fluorescence microscopy was developed to detect active yeast cells in cryosections of wheat dough. The sections were stained with 4',6-diamidino-2-phenylindole (DAPI) and counterstained with Evans blue. The active yeast cells in the sections appeared brilliant yellow and were readily distinguished from the red dough matrix. The dead cells allowed penetration of the Evans idlue through the cell membrane, which interfered with the DAPI staining and caused the dead cells to blend into the red environment. The number of active yeast cells in fermenting dough sections containing different proportions of living and dead yeast cells correlated well with the gas-forming capability of the yeast in the dough but not with the results of the conventional plate count method. The new method allows the study of yeast activity not only during the different stages of frozen dough processing but also during the fermentation of doughs.

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“…1. DAPI binds to DNA rich in adeninethymine (14,16). Only a few active cells are detectable by intense fluorescence.…”

Section: Resultsmentioning

confidence: 99%

Detection of Active Yeast Cells ( Saccharomyces cerevisiae ) in Frozen Dough Sections

Autio

Mattila‐Sandholm

1992

Appl Environ Microbiol

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“…For that, we determined 1) the number of nuclei per asexual spore and 2) the nuclear genotype of the asexual spores. For 1), asexual spores were scrapped off and immediately placed in 10 μl of the florescent dye DAPI, and we used fluorescence microscopy to visualise the nuclei ([ 46 , 47 ]; details in Additional file). For 2), 100 single spore cultures were genotyped.…”

Section: Methodsmentioning

confidence: 99%

The scope for nuclear selection within Termitomyces fungi associated with fungus-growing termites is limited

et al. 2014

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BackgroundWe investigate the scope for selection at the level of nuclei within fungal individuals (mycelia) of the mutualistic Termitomyces cultivated by fungus-growing termites. Whereas in most basidiomycete fungi the number and kind of nuclei is strictly regulated to be two per cell, in Termitomyces mycelia the number of nuclei per cell is highly variable. We hypothesised that natural selection on these fungi not only occurs between mycelia, but also at the level of nuclei within the mycelium. We test this hypothesis using in vitro tests with five nuclear haplotypes of a Termitomyces species.ResultsFirst, we studied the transition from a mixture of five homokaryons (mycelia with identical nuclei) each with a different nuclear haplotype to heterokaryons (mycelia with genetically different nuclei). In vitro cultivation of this mixture for multiple asexual transfers led to the formation of multiple heterokaryotic mycelia, and a reduction of mycelial diversity over time. All heterokaryotic mycelia contained exactly two types of nucleus. The success of a heterokaryon during in vitro cultivation was mainly determined by spore production and to a lesser extent by mycelial growth rate. Second, heterokaryons invariably produced more spores than homokaryons implying that homokaryons will be outcompeted. Third, no homokaryotic ‘escapes’ from a heterokaryon via the formation of homokaryotic spores were found, despite extensive spore genotyping. Fourth, in contrast to most studied basidiomycete fungi, in Termitomyces sp. no nuclear migration occurs during mating, limiting the scope for nuclear competition within the mycelium.ConclusionsOur experiments demonstrate that in this species of Termitomyces the scope for selection at the level of the nucleus within an established mycelium is limited. Although ‘mate choice’ of a particular nuclear haplotype is possible during mating, we infer that selection primarily occurs between mycelia with two types of nucleus (heterokaryons).

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“…It can also be used to determine degrees of ploidy within species, e.g., Schizosaccharomyces pombe (21), Phytophthora infestans (22-24), Itersonilia perplexans (2) and to compare DNA contents of different species belonging to the same genus (I1) or of species belonging to different genera (3,10,25).…”

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confidence: 99%

Quantitative differences of nuclear DNA contents and their taxonomic implications in Leucosporidium scottii, Rhodosporidium toruloides, and related basidiomycetous yeasts.

Suh

Kuroiwa

Sugiyama

1993

J. Gen. Appl. Microbiol.

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Estimates of the nuclear DNA (nDNA) content in the yeast cells of 43 strains of Leucosporidium scottii, Rhodosporidium toruloides, and related yeast taxa were analyzed by fluorescence microscope photometry. Quantitative differences of nDNA contents occur among 18 strains of L, scottii. Four groups of strains with similar nDNA content were recognizable in L. scottii. One group contained only one strain (IFO 9474) which was characterized by the Q-7 system. The respective strains of the remaining three groups had either Q-9 or Q-10 as the major ubiquinone system, except CBS 8188 having both of these. Some mating strains of L. scottii had almost twice the amount of nDNA as the presumed haploid value. In contrast, both mating type A and a strains of R. toruloides had nearly identical average nDNA contents. Our results suggest the presence of aneuploidy among yeast cells of L. scottii.Leucosporidium scottii is assigned to the teliospore-forming basidiomycetous yeasts (9). It has a multiple allelic bifactorial sexual incompatibility system, and mating is controlled by two factors designated A and B. Five A and three B factors have been identified (4). In addition, some strains are capable of homokaryotic formation of teliospores.Recently, a taxonomic revision of L. scottii has been required because of its heterogeneity of ubiquinone systems and low reproducibility of the mating reaction. Our recent studies (Joo et al. unpublished data) suggested that strains of L, scottii

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Cytofluorometric determination of relative DNA content in nuclei of Coniophoraceae (Boletales) using DAPI

Cited by 17 publications

References 15 publications

Detection of Active Yeast Cells ( Saccharomyces cerevisiae ) in Frozen Dough Sections

Detection of Active Yeast Cells ( Saccharomyces cerevisiae ) in Frozen Dough Sections

The scope for nuclear selection within Termitomyces fungi associated with fungus-growing termites is limited

Quantitative differences of nuclear DNA contents and their taxonomic implications in Leucosporidium scottii, Rhodosporidium toruloides, and related basidiomycetous yeasts.

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