An artifact in the alkaline hydrolysis of RNA labeled with guanosine-8-T

✍ Scribed by Fred H. Wilt

• Publisher: Elsevier Science
• Year: 1969
• Tongue: English
• Weight: 227 KB
• Volume: 27
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(69)90232-2

✦ Synopsis

An Artifact

in the Alkbline Hydrolysis of RNA Labeled with Guanosine-8-T1

We have employed guanosine labeled in the 8 position (guanosine-8-T) as a tracer for studying RNA synthesis in developing sea urchin embryos (1). When the distribution of H3 in the 2'(3') -mononucleotides formed during alkaline hydrolysis of RNA was studied, nucleotide label was found only in 2'(3')-guanylic acid (GMP), justifying the use of guanosine as a satisfactory tracer. However, a nonnucleotide component labeled with tritium was also evident when the hydrolyzate was fractionated on Dowex 50. This material is ~probably H3H0 formed by an exchange reaction under mild alkaline conditions between hydrogen isotope in the 8 position of the guanine base and the water. Proton exchange at the 8 carbon position of purines has been described (2-4) _ This is a practical consideration when using guanosine or guanine as a tracer in studies of RNA metabolism.

Methods. After exposure of embryos to 1 PC/ml of H3-guanosine (2700 mc/mmole, Nuclear Chicago), for 1 hr, sea urchin blastuIae were washed and frozen. RNA was prepared from the frozen pellet by a phenol method (5). The RNA was additionally treated with DNAse and pronase, and extracted with 2 M NaCl. All results obtained with this product can be repeated on mononucleotides resulting from a modified (6) Schmidt-Thannhauser fractionation of crude tissue fractions. Alkaline hydrolysis was carried out in sealed tubes in 0.3 iV KOH at 35°C for 16 hr. Mild acid hydrolysis was carried out in 1 N HClO, at 25' for 20 hr (7). Separation of 2'(3') -mononucleotides was carried out by the method of Katz and Comb (8) on Dowex 50 H+. Chromatographic