NUCLEIC ACIDS AND RELATED COMPOUNDS 24 1 



ISOLATION 



Both ribonucleic acid and deoxyribonucleic acid have been prepared from higher 

 plants. However, the latter is present in very low concentration in most plant tissues, 

 and successful isolations generally require as starting material a tissue in which nuclei 

 make up a large proportion of the cells. Wheat germ has been used for this purpose. 

 Many plant tissues contain reasonable amounts of RNA (e.g. 0.01% per weight of leaves), 

 and it may be prepared fairly readily. 



Thorough homogenization of the tissue is the first essential for extraction of either 

 type of nucleic acid. Older methods of preparing nucleic acid depended on extraction with 

 alkali but yielded a product which had suffered some degradation. Current methods use 

 extraction with neutral salt solutions and undoubtedly yield a less degraded product, but 

 in the absence of any absolute standard it cannot be claimed that the isolated nucleic acid 

 is obtained in its unchanged, native state. Isolation of nucleoprotein is even more open 

 to question. Products can readily be prepared which contain nucleic acid more or less 

 bound to protein, but in the absence of any standards they may be regarded as anything 

 from the intact nucleoprotein as it exists in the cell to nucleic acid contaminated with ex- 

 traneous protein. 



The four types of non-dialyzable, water-soluble substances likely to be encountered 

 in tissue extracts are polysaccharides, proteins, ribonucleic acids, and deoxyribonucleic 

 acid. The rationale for separating the nucleic acids is generally based on denaturing and 

 coagulating proteins by high speed homogenization in the presence of chloroform-octyl 

 alcohol (8: 1). Nucleic acids may then be freed of polysaccharides by making the solution 

 weakly acidic to precipitate the nucleic acid or basic to dissolve the nucleic acid and leave 

 polysaccharides as an insoluble residue. Dialysis removes any small molecules. 



DNA is separated from RNA by the choice of original extraction medium. DNA is 

 more tightly bound to cell structure than is RNA. The latter is solubilized by homogeniza- 

 tion with dilute (e.g. 0. 1 M NaCl). After removal of this supernatant solution, DNA may 

 be extracted with 1-3 M NaCl and re -precipitated by diluting to 0. 1 M NaCl. The chief 

 drawback to this extraction procedure is the presence of enzymes which may degrade the 

 nucleic acids. Enzymes may be inactivated by first dropping the tissue into boiling ethanol, 

 which does not dissolve nucleic acid. Tissues containing much lipid or fat -soluble pigment 

 should also be defatted before extraction. The references (15-18) may be consulted for 

 additional details of procedure. It must always be kept in mind that techniques developed 

 for animal tissues may have to be modified for use with plants. 



Isolation of nucleotides from plants has been greatly aided by ion exchange chroma- 

 tography. Initial extraction of the tissue is usually carried out with 10% trichloracetic or 

 perchloric acid so that proteins and nucleic acids are left with the residue. Further steps 

 often depend on precipitating phosphates as barium salts. Careful control of pH is impor- 

 tant at this stage since barium salts of nucleotides tend to be more soluble than the barium 

 salt of phytic acid, a common constituent of seedlings. Thus at pH 4 barium phytate is 

 insoluble and the barium salt of ATP remains in solution (19). Further separation and 

 purification of the various nucleotides is most conveniently performed by chromatography 

 over anion exchange resin such as Dowex 1. Chromatography on charcoal columns may 

 also be useful and is commonly used for purification of DPN (NAD) and TPN (NADP) (20). 



Nucleosides and free nitrogen bases are also extracted with dilute acid from plant 

 tissues. It is hard to make any generalizations concerning methods of purifying them. 

 A few techniques can be mentioned, but there are rather wide differences in solubility 

 and reactivity so that the general references should be consulted for details. The free 

 bases and their nucleosides are often precipitated as complex mercury salts and then 



