Isolation and Characterization of Chloroplast DNA from the Marine Chromophyte, Olisthodiscus luteus: Electron Microscopic Visualization of Isomeric Molecular Forms

Abstract

Chloroplast DNA (ctDNA) from the marine chromophytic alga, Olisthodiscus luteus, has been isolated using a whole cell lysis method followed by CsCl-Hoechst 33258 dye gradient centrifugation. This DNA, which has a buoyant density of 1.691 grams per cubic centimeter was identified as plastidic in origin by enrichment experiments. Inclusion of the nuclease inhibitor aurintricarboxylic acid in all lysis buffers was mandatory for isolation of high molecular weight DNA. Long linear molecules (40 to 48 micrometers) with considerable internal organization comprised the majority of the ctDNA isolated, whereas supertwisted ctDNA and open circular molecules averaging 46 micrometers were occasionally present. Also observed in this study were folded ctDNA molecules with electron dense centers ("rosettes") and plastid DNA molecules which have a tightly wound "key-ring" center. The ctDNA of Olisthodiscus has a contour length that is median to the size range reported for chlorophytic plants.A minor component of the total cellular DNA, which originates from a DNase insensitive cellular structure, has a buoyant density of 1.694 grams per cubic centimeter. This DNA consists predominantly of linear molecules, but open circles 11.5 micrometers in length and rare 22-micrometer dimers were also present.This study represents the first analysis of the extranuclear DNA of a chromophytic alga.