Use of the fluorochrome 4'6-diamidino-2-phenylindole in genetic and developmental studies of chloroplast DNA.

Abstract

Use of the DNA-specific fluorochrome 4''-6-diamidino-2-phenylindole (DAPI) makes it possible to examine in situ the structure of chloroplast DNA (chDNA) with the fluorescence microscope. This simplifies the study of genetic and developmental changes in chloroplast DNA. Three examples are presented. Wild type Euglena gracilis B contains several chloroplast DNA nucleoids per chloroplast. A yellow mutant lacking functional chloroplasts is similar, but such nucleoids are absent in an aplastidic mutant strain known from biochemical studies to have lost its chDNA. In vegetative cells of the giant-celled marine algae Acetabularia [A. calyculus and A. crenulata] and Batophora [B. oerstedii], only about 1/4 of the chloroplasts have even 1 discernible chloroplast DNA particle; such particles vary in size, showing a 30-fold variation in the amount of DNA-bound DAPI fluorescence detected per chloroplast. Of chloroplasts in developing Acetabularia cysts 98% contain chDNA, with as many as 9 nucleoids per chloroplast. DAPI-stained chloroplasts of chromophyte algae [Ditylium brightwellii, Coduim fragile and Sphacelaria sp.] display the peripheral ring of DNA expected from EM studies. The rings are not uniform in thickness but are necklace-like, with the appearance of beads on a string. The multiple nucleoids in plastids of chlorophyte algae also appear to be interconnected throughout the chloroplast; a common structural plan may underlie chDNA morphology in both groups of algae.