Fig. 1

(a). Separation of purines and ribonucleoside monophosphates by reverse phase HPLC. Curve (a), adenine; curve (b), guanine; curve (c), mixture of adenine, guanine, adenosine 2’-phosphate and adenosine 3’-phosphate; curve (d), mixture of adenosine 2’-phosphate and adenosine 3’-phosphate. (b). Release of adenine from pure DNA by acid hydrolysis. The DNA was incubated in 0.15 N or 0.20 N HCl at 60 °C for 20, 40 or 60 min, then the amount of released adenine was quantified by HPLC. The quantity of DNA (calculated from the amount of adenine) was compared to the amount specified by the manufacturer (Novagen). For the 0.15 N treatment (solid circle), the amounts were 34.3, 86.7 and 94.5 %, respectively. For the 0.20 N treatment (solid square), the amounts were 99.3, 103.3 and 103.3 %, respectively. Error bars represent range of duplicate samples; where not shown, the values are within the symbol. (c). Selective release of adenine from DNA compared to RNA. DNA (curve a), RNA (curve b) and poly(rA) (curve c), were subjected to standard acid/alkali treatment, then analysed by HPLC. The expected peaks of guanine and adenine from DNA, adenosine 3’-phosphate and adenosine 2’-phosphate from poly(rA) and adenosine 3’-phosphate, adenosine 2’-phosphate and guanosine 2’-phosphate from RNA can be seen. In addition, tiny peaks of adenine can be seen in the poly(rA) and RNA samples, representing 0.25 and 1.63 % of the total adenosine phosphate from poly(A) and yeast RNA, respectively. Peaks in this and subsequent figures are identified by the following numbers: 1, adenosine 5’-phosphate; 2, guanine; 3, guanosine phosphate; 4, adenosine 3’-phosphate; 5, adenine; 6, adenosine 2’-phosphate. The guanosine phosphate isomer (Peak 3) is likely to be the 2’-phosphate, based on the order of elution of the adenosine monophosphates in our isocratic HPLC system and of the guanosine and adenosine phosphates on an ion-exchange system [22]. Absorbance at 260 nm in milliabsorbance units; adenine (AUC), Area Under the Curve for adenine in milliabsorbance units