Tests which distinguish induced crossing-over and aneuploidy from secondary segregation in Aspergillus treated with chloral hydrate or gamma-rays

Abstract

A system of tests with the ascomycete Aspergillus nidulans was devised that can detect 3 primary effects of genotoxic agents: (1) increases in mitotic crossing-over; (2) induced aneuploidy; and (3) clastogenic effects which cause chromosomal imbalance. Conidia of a new diploid tester strain, heterozygous for 4 recessive markers which alter conidial color, are treated and plated onto nonselective media. In cases of induced crossing-over, large color segments are found in normal green colonies, frequently adjacent to reciprocal twin segments. In contrast, both malsegregation and chromosome breakage produce unbalanced types which grow poorly and segregate further. Cases with yellow segregants are replated and their secondary diploid sectors tested for markers which are located on both chromosome arms in coupling with yA. Induced aneuploidy can be distinguished from chromosome breakage by the pattern of marker segregation. Any aneuploid type will produce euploid sectors solely by segregation of whole chromosomes; trisomic colonies (yA / yA / +) will show 1:2 ratios for yellow (homozygous yA) to parental green (yA/+) sectors and have characteristic phenotypes. Other induced unbalanced types, if heterozygous for deletions or aberrations may produce yellow diploid sectors by secondary crossing-over as well as by nondisjunction and such cases show unique patterns of genetic segregation and non- predictable phenotypes. As a complementary test, haploid strains are treated and induced abnormally growing types are replated and classified by phenotype. Aneuploids are unstable and produce many normal sectors, and some of these disomic or trisomic types can be visually identified.In contrast, induced deletions are lethal, and duplications or 'morphological' mutants show much more stable abnormal phenotypes. This test system was used to characterize the primary effects of gamma-rays and chloral hydrate. Results and evidence were as follows: (1) A dose-dependent increase of color segments resulting from reciprocal crossing-over was found after treatment of dividing nuclei in germinating diploid conidia with gamma-rays, but not with chloral hydrate. (2) Highly aneuploid and polyploid types were induced in diploid and haploid germinating conidia by chloral hydrate but not to any significant extent by gamma-rays. (3) gamma-Rays caused a dose- dependent increase off abnormally growing colonies when dormant or germinating diploid conidia were treated. These colonies produced secondary euploid sectors by spontaneous nondisjunction and frequently also by crossing-over, which provided evidence for induced semidominant and recessive lethal mutations of many types.