Summinary. Radioautographic and radiochemical techniques were used to establish the presence of replicating DNA in the chloroplasts of Acetabutlaria miediterranea. These techniques also demonstrated that these chloroplasts synthesize RNA. It was found that label from thymine was also incorporated into DNA and RNA in these chloroplasts.With the establishment of protein and nucleic acid synthesis in Acetabutlaria chloroplasts, it is clear that these chloroplasts carry out those metabolic processes which are most characteristic of autonomous cells.To demonstrate the presence and/or synthesis of DNA in chloroplasts, it is of the utmost importance to be able to eliminate completely any possibility of nuclear DNA's interferring with the determination.Acctabularia is the ideal organism for these studies:it can easily be enucleated, its cells live for several months in this enucleated state, and its size and growth habit make it ideally suited for the various manipulations required in the experiments.Primiary emphasis in studying chloroplast DNA has been placed on establishing the presence of the DNA in chloroplast preparations (2,5,6,7,9,11,18,20).There are many characteristics of chloroplasts that would lead one to expect that this DNA is an active DNA that is replicating and synthesizing RNA (22). For example, it has been recognized for some time that chloroplasts are capable of growing and dividing independently of the growth and division of the cell. Nitella chloroplasts demonstrate this clearly (10) and in Acetabtlaria, the chloroplasts grow and divide in enucleated cells.The presence of replicating DNA in chloroplasts was investigated using thymidine-3H incorporation in Acetabdlaria (3), Clivia (16) (2) to prove that these chloroplasts do contain DNA.While thymidine is readily incorporated into DNA and is usually used in tracer studies concerning DNA (1,8,15), the free base thymine is not well utilized (14,15,17). It has been noted that thymine is incorporated into a nucleic acid fraction in Acctabuilaria (3).It was the purpose of this study to establish conclusively the synthesis of DNA and RNA by chloroplasts of Acetabularia miediterranea.
Materials and MethodsExperimental Organism,. Acetabtularia inediterraica (Lamoiuroux) cultures were growni in artificial sea water medium according to the methods of Keck (12) as modified by Schreiber et al. (21).Preparationt of Acetabularia Cells for Radioaittography. Nucleated and enucleated cells of Acetabutlaria were placed in 10 ml of medium containiing 25 1uc of either thymidine-methyl-3H or thyminemethyl-3H (6.0 c/mmole). After varying incubation times, the cytoplasm was transferred to microscope slides by squeezing the cytoplasm from the cells with tweezers and smearinig the drop on the slide. The slides were waslhed with cold 12 % trichloroacetic acid for 10 mintutes, tlheni with distilled water for 5 minutes. Somiie slides were theni treated with deoxyribonuclease (3 nig/mIl, with 0.001 M Mg1,SO4 a(l(le(l) for 3 hoturs at room temperature -and( waslhed with trichlor...