A closed-circular DNA duplex, especially a plasmid, for example, is known to have a n interwound right-handed (negative sense by definition) superhelical structure when it is isolated "intact" from a living cell.' Such a superhelical structure is considered to involve appreciable alterations in DNA secondary structure2 from a n ordinary double-helical structure. A local denaturation in such a DNA was suggested, for example, by its enhanced susceptibility to S1 nuclease 3-5 and its reduced susceptibility to EcoRl nuclease.6 In an attempt to find a local structural change associated with such a "denaturation," we have examined its Raman spectrum.
We used a plasmid, pFb100, with 5451 base pairs, which was derived from pBR322 with a fibroin gene? A 1.4-kbp fibroin DNA fragment was subcloned into the larger fragment of the pBR322/HindIII/BamHI digest? This plasmid sample was prepared through its transfection into Escherichia coli HB101, the selection of ampicillin-resistant colonies, a culture with chloramphenycol amplification, and an extraction of DNA from the harvested cells. The bacteria cells were lysed by a treatment with lysozyme and a detergent mixture of Brij 58 and sodium deoxycholate, and the plasmid DNA was separated from the bacterial DNA by 15,000 rpm centrifugation8 The plasmid DNA was extracted with phenol, and the closed-circular DNA (pFb100-I) isolated by ethidium bromide-CsCl bouyant density-gradient centrifugation. An RNase treatment was then performed on the isolated DNA sample for some contamination of RNA, and DNA was extracted with a n organic solvent mixture of phenol, CHCl,, and isoamylalcohol (50:50:1). Its purity was confirmed by a n electrophoresis map of its fragments with restriction enzymes.
The sample for the Raman spectroscopy was obtained through dialysis against 10 m M NaCl aqueous solution and its lyophilization. This was then dissolved to form a 2% solution in 0.085MNaCl + 0.2 mMEDTA, pH 7.0 in a capillary.
The tertiary structure of the DNA sample in this study was monitored by agarose gel electrophoresis (see Fig. ). Immediately after placing the DNA sample in the capillary tube, 85% of it showed the mobility of form I, which is generally understood to be a n interwound right-handed superhelix with a superhelical density of 1/100 -1/300 bp? It has been found, however, that it contains about 15% nicked plasmid (form 111, which has a lower mobility. A cleaved, linear plasmid (form 1111, however, should have a slightly higher mobility (Fig. , lanes b and k), and our sample has been proved not to involve it. In addition, the 514.5-nm beam (100 mW at the sample point) from a n NEC