376 MAHLON B. HOAGLAND 



All of this work is in its infancy but a few of the preliminary approaches to 

 the problem may be outlined. 



The most direct means of separating and purifying transfer RNA is to 

 centrifuge at 100,000 g for a longer period, since transfer RNA is of rela- 

 tively low molecular weight. Early experiments indicated that a centrifuga- 

 tion at 100,000 g for 3 hours instead of 1 hour reduced the RNA content of 

 the supernatant fraction by 50% with no loss in amino acid acceptor- 

 ability. 117 It is likely that the material sedimented during the prolonged 

 centrifugation was microsomal in nature. Salt fractionation, such as that 

 used by Davis and Allen, 139 may prove to be effective in removing higher 

 molecular weight components. ECTEOLA has been reported 117 to give 

 some separation of transfer RNA from inactive RNA. 



With respect to the separation of transfer RNA's one from the other, 

 Smith et al. m have effected some preliminary fractionation with the use of 

 calcium phosphate gel. Another approach to transfer RNA fractionation 

 has been the use of countercurrent distribution as reported by Holley and 

 Merrill. 142 These workers have obtained partial separation of the transfer 

 RNA which accepts alanine from other transfer RNA's by this means. 



Because of the relatively high molecular weight of the specific transfer 

 RNA molecules and the probability that the molecular weight distribution 

 is narrow, fractionation based on size, electrical charge, solubility, difference 

 in base content, etc. may prove to be difficult. For this reason several labora- 

 tories have attempted to "pull out" a particular transfer RNA by making 

 use of the chemical properties of the amino acid it specifically binds. Pre- 

 liminary success has been reported by Brown 143 who has been able to iso- 

 late selectively the tyrosine and histidine transfer RNA's by binding the 

 amino acid moiety to a diazotized polydiazostyrene column. A similar ap- 

 proach is to make use of the fact that the amino acid protects its particular 

 RNA from oxidation of the 2'- and 3'-hydroxyl groups of the terminal ri- 

 bose residue by periodate. The oxidized terminal aldehydic groups could 

 then be utilized for addition reactions to produce RNA molecules suffi- 

 ciently different from the unaltered ones to permit separation. 96 



b. Reactions Involving the Terminal Nucleotides of Transfer RNA 



(1) Early Studies on Nucleotide Labeling of Soluble RNA and Terminal 

 Attachment of Nucleotides. Studies in cell-free systems on incorporation of 

 RNA precursors into RNA date back to 1949. 144 The general purpose of 



141 K. C. Smith, E. Cordes, and R. S. Schweet, Biochim. el Biophys. Acta 33, 286 

 (1959). 



142 R. W. Holley and S. H. Merrill, Federation Proc. 18, 982 (1959). 



143 G. Brown, A. V. W. Brown, and J. Gordon, Brookhaven Symposia in Biol. 12, 47 

 (1959). 



'« M. Friedkin and A. L. Lehninger, J. Biol. Chem. 177, 775 (1949). 



