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Nucleic Acids Research, 1980, Vol. 8, No. 15 3371-3391
© 1980

MOLECULAR BIOLOGY

Saccharomyces cerevisiae plasmid, Scp or 2 urn: intraceDular distribution, stability and nucleo-somal-like packaging

V.L. Seligy, D.Y. Thomas and B.L.A. Miki

Molecular Genetics Group, Division of Biological Sciences, National Research Council Ottawa, K1A0R6, Canada

Received March 13, 1980. Cell fractions from yeast strains known to harbor the plasmid 2 µm or Scp were treated with nucleases used to probe eukaryotic chromosome structure. Scp and subfragments were identified by hybridization to natural or cloned Scp probes according to Southern (34). Specificity was confirmed with non-Scp probes. Copy/haploid nuclear genome(n) was estimated from reconstructions at a resolution of 0.5/n. About 43–67% of the total cellular copy exists as nucleoprotein complexes which separate from other debris on i isokinetic sucrose gradients with s-values of 67–110. These complexes are totally degraded by DNAase I. Digestion with micrococcal nuclease produced integral-sized fragments; they are not generated by direct mixing of pure Scp with nuclear chromatin from a[cir°] strain. Initial digests gave a repeat of 168±3 base pairs (bp) for both Scp and nuclear nucleoprotein; advanced digests reduced the nuclear repeat relative to Scp by {small tilde}8 bp. Of a potential 37 repeat units/plasm1d, 31–32 were directly measured. A strain difference in Scp autodegradation was found. A partial nuclease resistant form was also demonstrated whose abundance was cell strain and growth stage dependent. Both Scp isomers exist in these complexes which are structurally similar to simian viral 40 minichromosomes.


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