Two‐dimensional gel analysis of repetitive nuclear DNA sequences in the genome of Physarum polycephalum

Abstract

1. The composition of repetitive sequences in restriction patterns of nuclear DNA of Physarum polycephalum was determined by high-resolution gel analysis.2. Three types of repeated DNA fragments in the size range of (0.2-2) x lo3 base pairs could be identified as discrete spots on the gels and distinguished by their abundance and above-average base composition of either guanine and cytosine (G + C) or adenine and thymidine (A +T). 3.On comparing the DNA composition from exponentially growing plasmodia with that of starved plasmodia, which have become competent to sporulate and have lost 80% of their nuclei, no change was detected among the (A + T)-rich repeat fractions, whereas several of the (G + C)-rich fractions revealed fewer copies in the DNA prepared from starved cells.4. As shown by hybridization under saturating conditions, the reduction of several (G + C)-rich repeated sequences in the restricted nuclear DNA in sporulation-competent cells can be explained by a 64% elimination of the extrachromosomal nucleolar ribosomal DNA sequences.The DNA content in the slime mold Physarum polycephalum is extraordinarily variable. Different strains contain different amounts of nuclear DNA and there exist constitutively mixoploid strains, like M3cIV with two kinds of nuclei differing by 18% in their DNA content [l]. Furthermore, the nuclear DNA content has been shown to decrease as a function of aging by about 20% in M3cVII [2].It is not known whether the variable DNA contents in Physarurn reflect overreplication and underreplication of genomic segments in response to physiological conditions, or the establishment of a germline during sporulation.We assume that expansion and diminution of the genome of Physarurn should affect the composition of repetitive sequences. These are non-randomly distributed over the genome in Physarum During starvation, surface plasmodia (macroplasmodia) of Physarum degrade a large part ( x 80%) of their nuclei as well as RNA, proteins and glycogen, reaching a state of competence to sporulate after 3 -4 days. Sporulation can then be induced by illumination, which initates the irreversible sporulation pathway. During this differentiation process most of the remaining 20% of the nuclei are packed up into spores, where they undergo meiosis [7].We have compared the restriction patterns of the nuclear DNA from plasmodia growing exponentially in liquid culture (microplasmodia) and from starved sporulation-competent plasmodia in order to identify a possible germline-specific composition of repetitive sequences. The restriction patterns were analyzed two-dimensionally, which increases the resolution and simultaneously gives rapid information about the relative G + C or A + T content of the individual fragments The only repetitive sequences for which we could demonstrate a change in frequency are the extrachromosomal nucleolar sequences, which were reduced to about 36% of the control value in the plasmodia competent for sporulation.The Physarum nucleolar DNA molecules are extrachromosomal, autonomousl...