Development and standardization of a mutagenicity test system using Aspergillus nidulans as test organism

J.G. Boschloo

Research output: Thesisexternal PhD, WU

Abstract

The suitability of Aspergillusnidulans as a test organism for mutagenicity testing has been investigated. Several test systems have been tried. Those systems were preferred in which the mutants or recombinants could be selected without the need of adding a compound to the medium which itself is genetically active.

Four incubation methods have been compared. The plate incorporation assay (method 1) is the easiest method for testing chemical compounds, but it is often difficult to obtain reliable quantitative data. Moreover, complications may arise when the compound is toxic or causes a selective advantage of the mutants. In the liquid suspension test (method 2) the conditions can be controlled more accurately, so this method is more suitable for obtaining reliable quantitative data. For testing germinating conidia, the suspension test (method 3) turned out to be less suitable, since some conidia germinated directly from the start causing - in spite of the presence of 0.2% agar - big clumps of conidia at the end of the incubation period. Germinating conidia (and growing mycelium) can better be tested with the media mediated assay (method 4). However, an extra control experiment must be performed in order to discriminate between a real mutagenic activity and a selective advantage.

Strain 110, which carries the methGl allele, proved to be very suitable for measuring the point mutation frequency when methionine independent suppressor mutants are scored. The instability of a duplication strain can be also used as an indication for the genetic activity of a chemical compound. The construction of a duplication strain heterozygous for the recessive sorA2 allele (strain 007) reduced the number of plates needed for the scoring of segregants. The exact process by which the sectors arise is not known. Some crossing-over products are scored as deletion products when no special attention is
paid to the morphology of the segregants.
For determining the crossing-over and non-disjunction frequencies scoring of mitotic segregation is the easiest way, at least when it is not intended to distinguish the inducing processes. If such a distinction is required, it is easier to select the recombinants. Strain D4 can be used to select recombinants homozygous for the suAladE20 allele, and the crossing-over and non-disjunction products can be directly distinguished by their conidial colour. In practice, complications arose since the conidial colour was faint and the leaky growth of the test strain led to a concentration effect. Selection of sorbose resistant recombinants was even less suitable, and a combinated selection of sorbose resistant and adenine prototroph recombinants proved unfeasible. Selection of pimaricin resistant recombinants of strain D7 was much easier and therefore preferably used in this study. However, since pimaricin itself is known to induce non-disjunctions this system has only been used in combination with the media mediated assay to minimize the risk of complications.

The system for measuring the frequency of recessive lethals developed by Morpurgo et al. (1978) has also been investigated, but the background in the control was relatively high. This was most likely caused by incomplete haploidization on FPA medium. Changing the medium did hardly improve the results. This implies that one can only measure larger effects on the frequency of recessive lethals.
Several chemicals, mostly with known mutagenic properties have been tested by means of the proposed test systems. These confirmed the suitability of the test systems.
Thus, in addition to other mutagenicity tests like the Ames test with bacteria, the fungus Aspergillusnidulans offers good opportunities to asses the genetic activity of chemical compounds on eucaryotic organisms.

Original languageDutch
QualificationDoctor of Philosophy
Awarding Institution
Supervisors/Advisors
  • van der Veen, J.H., Promotor, External person
Award date29 May 1985
Place of PublicationWageningen
Publisher
Publication statusPublished - 1985

Keywords

  • aspergillus
  • biological techniques
  • equipment
  • experiments
  • methodology
  • mutagenesis

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