280 J. D. SMITH 



corresponding nucleosides produced during the BaCOs hydrolysis. After 

 elution from the bands the cyclic nucleotides are isolated by electrophoresis 

 at pH 3.5, when they run in the positions of the corresponding noncyclic 

 nucleotides. 



As they bear no secondary phosphate group, at pH 7.4 the cyclic nucleo- 

 tides carry only one negative charge, while other nucleotides carry two 

 (Figure 1). This gives a simple electrophoretic method of separating cyclic 

 nucleotides from other mononucleotides. 



b. Polynucleotides from Ribonucleic Acid 



With the exception of the very small amounts of polynucleotides orig- 

 inating from end groups the polynucleotides in complete ribonuclease 

 digests are of one type.^' ^^'^^ They consist of chains of one or more purine 

 nucleoside residues joined through 3',5'-phosphodiester links, and ter- 

 minated by a pyrimidine nucleoside residue which is joined through a 

 phosphodiester link on its 5 '(OH)- to the 3'- position on the adjacent purine 

 nucleoside residue, and which bears a singly esterified phosphate in the 

 3'-position. These are thus of the type, AC, AU, AGC, etc. In partial di- 

 gests of PNA are also found the corresponding polynucleotides bearing 

 cyclic (2',3'-monohydrogen phosphate) groups on the terminal pyrimidine 

 nucleoside residue, AC!, AGC!, etc. The partial digests also contain small 

 amounts of pyrimidine polynucleotides with cyclic end groups such as 

 UU!, UUU!, CC!, etc. 



The movement of these substances on a paper chromatogram in solvent 

 1 largely depends on three factors, namely, (1) mononucleotides move 

 faster than the corresponding dinucleotides, and dinucleotides move faster 

 than trinucleotides; (2) substances with cyclic terminal phosphate groups 

 move faster than the corresponding substances with singly esterified ter- 

 minal phosphates ; (3) guanylic acid and its derivatives move more slowly 

 than the corresponging mono- and polynucleotides. Chromatography in 

 this solvent thus fractionates ribonuclease digests into a number of bands 

 from which many individual polynucleotides may be separated by single 

 electrophoretic runs at pH 3.5. The chromatographic separation and electro- 

 phoretic mobilities of these are illustrated in Table II. Often more than 

 one electrophoretic run is necessary to separate the substances in a single 

 chromatographic band. For example band 1 of such a chromatogram eluted 

 and subjected to electrophoresis at pH 3.5 for 2 hr. at 20 v. /cm. gives a 

 band moving 13.5 cm. (towards the anode). This is a mixture of GC and 

 AAC which may be resolved by eluting and either (1) running at pH 5, 

 when the trinucleotide moves faster than the dinucleotide, or (2) running 

 at pH 2.5 for 3 hr. at 16 v. /cm., when GC moves 4.8 cm. and AAC 6.4 cm. 



