Orthogonal-field Electrophoresis Research Articles

Electrophoretic karyotype analysis of the button mushroom, Agaricus bisporus

An efficient procedure is presented for the generation of protoplasts from shake flask cultures of the commercial mushroom Agaricus bisporus. Orthogonal field electrophoresis (OFAGE) of high molecular weight DNA from lysed protoplasts resolved the genome of A. bisporus into at least 10 bands, ranging in size from 1.2 to 4 Mb. The illustrated electrophoretic karyotypes of two homokaryons were highly polymorphic. A heterokaryon of the two homokaryons contained a mixture of the two electrophoretic patterns, though the ratio of nuclear types was not equal. A number of RFLP and RAPD markers and the rDNA repeat have been localized to specific chromosomes. Based on ethidium bromide staining intensity and autoradiography, we have estimated the chromosome number of A. bisporus to be 13.Key words: Agaricus bisporus, orthogonal field electrophoresis, RFLP, rDNA.

Tandem repeats in extrachromosomal ribosomal DNA of Dictyostelium discoideum, resulting from chromosomal mutations.

Extrachromosomal ribosomal DNA in the simple eukaryote Dictyostelium discoideum is readily separated from chromosomal DNA by orthogonal field electrophoresis (OFAGE), forming a prominent band in the 110-kb region of the gel. Here we show that mutations in at least two chromosomal genes give rise to a ladder of rDNA bands increasing in size up to about 300 kb. One of these mutations, the rrcA350 allele, which is recessive to wild type and maps to the centromere-proximal region of linkage group II, has an unstable phenotype; spontaneous revertants, which no longer exhibit the rDNA ladder, have been recovered. Another mutation rrc-351, provisionally mapped to linkage group IV, is dominant to wild type. The rDNA ladder is caused by concatamerization of a 34-kb fragment in the nontranscribed central spacer region of the 88-kb linear rDNA palindrome. Restriction enzyme analysis has revealed that each concatamer is generated by crossovers between two rDNA molecules.