Dictyostelium discoideum: A Valuable Eukaryotic System for Repair Studies

Journal of General Microbiology

1976

Self Cite

The linkage of two mutations leading to increased sensitivity to ultraviolet light and 60Co gamma rays was determined in the slime mould Dictyostelium discoideum using a genetic analysis based on the parasexual cycle. Diploids were selected from a mixture of radiation-sensitive, temperature-resistant and radiation-resistant, temperature-sensitive haploids on the basis of simultaneous radiation and temperature resistance. Analysis of drug-resistant haploid segregants of the heterozygous diploids indicated that one of the radiation-sensitive mutations, radA20, was linked to linkage group I whereas the other, radB13, was linked to the recently defined linkage group VI.

Section: Introductionmentioning

confidence: 99%

Genetic Analysis of Radiation-sensitive Mutations in the Slime Mould Dictyostelium discoideum

Welker

Journal of General Microbiology

1976

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“…The cellular slime mold Dictyostelium discoideum is a useful organism for the study of deoxyribonucleic acid (DNA) repair in replicating vegetative and nonreplicating developing cells (3). The normally haploid life cycle of this organism has facilitated the isolation of radiationsensitive mutants which are impaired in their ability to repair damaged DNA (5).…”

mentioning

confidence: 99%

Excision of pyrimidine dimers from nuclear deoxyribonucleic acid in ultraviolet-irradiated Dictyostelium discoideum.

Clark

1981

Mol. Cell. Biol.

A sensitive endonuclease assay was used to study the fate of pyrimidine dimers introduced by ultraviolet irradiation into the nuclear deoxyribonucleic acid of the cellular slime mold Dictyostelium discoideum. Analysis of the frequency of T4 endonuclease V-induced single-strand breaks by alkaline sucrose gradient sedimentation showed that strain NC4 (radt) removed >98% of the dimers induced by irradiation at 40 J/m2 (254 nm) within 215 min after irradiation. HPS104 (radC44), a mutant sensitive to ultraviolet irradiation, removed 91% under these conditions, although at a significantly slower rate than NC4: only 8% were removed during the 10-to 15-min period immediately after irradiation, whereas NC4 excised 64% during this interval. HPS104 thus appears to be deficient in the activity(ies) responsible for rapidly incising ultraviolet-irradiated nuclear deoxyribonucleic acid at the sites of pyrimidine dimers.The cellular slime mold Dictyostelium discoideum is a useful organism for the study of deoxyribonucleic acid (DNA) repair in replicating vegetative and nonreplicating developing cells (3). The normally haploid life cycle of this organism has facilitated the isolation of radiationsensitive mutants which are impaired in their ability to repair damaged DNA (5). These mutations can be genetically mapped, and the molecular basis for their radiation-sensitive phenotypes can be investigated (4,12,15). Linkage and complementation studies have to date identified nine loci (radLA to radI) which confer sensitivity to 6sCo gamma rays or 254-nm ultraviolet (UV) light or both (2,25). Mutations at the radA and radC loci show a synergistic response to UV irradiation when combined in double-mutant haploid strains, indicating that these loci participate in separate pathways for repair of UV damage. The radB locus operates in the same repair pathway as radA since radA-radB double-mutant haploids are no more sensitive to UV than is radB, the more sensitive single mutant (27). Mutants in all complementation groups except radC make and rejoin singlestrand breaks in their DNA after UV irradiation; radC mutants are apparently deficient in an endonuclease activity specific for UV-irradiated DNA (26). Using the pyrimidine dimer-specific T4 endonuclease (endo V) to assay for the presence of these lesions in DNA (11), we have further characterized the dimer excision capabilities of UV-resistant and -sensitive strains of D. discoideum. Growth and labeling of D. discoideum strains. Strains NC4 (wild type) and HPS104 (radC44 axeAl axeBI tsgAl) were routinely grown on standard agar plates in association with E. coli B/r as previously described (5). 3H labeling of D. discoideum DNA was carried out essentially as described by Welker and Deering (26). 14C labeling of D. discoideum DNA was carried out in an analogous manner by growing slime mold cells on E. coli 15T whose DNA had been specifically labeled during prior growth on C-rninimal medium supplemented with 2.0 Ag of [2-14C]thymine per ml at a final specific activity of 0.06 Ci/mmol (Amersh...

In vivo nicking and rejoining of nuclear DNA in ultraviolet-irradiated radiation-resistant and sensitive strains of Dictyostelium discoideum

Welker

1979

Molec. Gen. Genet.

Some aspects of DNA repair in several radiation-resistant and radiation-sensitive strains of Dictyostelium discoideum were investigated by using alkaline sucrose gradients to analyze for the production and resealing of single-strand breaks following irradiation with 254 nm UV. All radiation-resistant strains and all mutants assayed that are sensitive to both UV and 60Co gamma rays produced single-strand breaks in their nuclear DNA after a UV fluence of 15 M/m2. Mutants at the radC locus which are sensitive to UV but as resistant as their parental strains to 60Co gamma rays produced many fewer single-strand breaks in their DNA after irradiation with UV. Thus, the radC mutations alter a repair pathway specific for UV-induced DNA damage and presumably affect the activity of a UV-damage-specific endonuclease involved in excision repair. All radiation-resistant strains and all of our mutants sensitive to gamma rays rejoined much of their DNA during a three-hour post-UV-irradiation incubation, suggesting that these strains have at least a partially intact excision repair system.