34. THE RIBONUCLEIC ACIDS OF VIRUSES 263 



the enzyme. Since the splitting of RNA, as well as the inactivation, starts 

 without delay upon addition of ribonuclease, both processes can be con- 

 sidered to be single-hit. If the enzymic splitting of the RNA backbone 

 occurs statistically with a probability (a) per bond and per unit of time, 

 and if the biologically active unit consists of N such bonds, the decrease in 

 infectivity (/) with time (t) may be represented by an equation 



In (///o) = Nat (3) 



(with I = infectivity at time t = 0, 1 = infectivity at time t). When one 

 internucleotide bond has been broken per infective unit by the action of 

 ribonuclease, the infectivity is reduced by a factor e. The molecular weight 

 can be calculated if the activity of the enzyme, expressed as the propor- 

 tions of bonds split per unit of enzyme concentration and time, is meas- 

 ured under the conditions used for the infectivity test. 



The enzymic activity can be determined by the titration of acid phos- 

 phate groups released upon splitting, as well as by the increase in UV ab- 

 sorption at 260 niju which is always observed upon degradation of RXA 

 molecules. Empirically, it was found that the appearance of one acid group 

 per 22 nucleotides (molecular weight = 22 X 320 = 7000) corresponds to 

 an increase in UV absorption of 15%. In order to obtain an equivalent 

 increase in absorption in the time in which the infectivity decreases to a 

 value \/e, it is necessary to increase the ribonuclease concentration 260 

 times of that necessary for the measured decrease in infectivity. From 

 these data, Gierer 11 calculated the number of bonds or number of nucleo- 

 tides as 5700, corresponding to a molecular weight for the infectious RXA 

 molecule of 5700 X 320 = 1.8 X 10 6 . 



Thus, it was possible, using quite a variety of techniques, to ascribe a 

 molecular weight of approximately 2 X 10 6 to the infectious RNA within 

 the limited accuracy of the determinations. This value corresponds to the 

 total RNA content of the virus particle. The genetically active substance 

 seems to be associated with approximately 6000 nucleotides which are 

 bound to each other. The experiments presented, so far, do not exclude the 

 possibility that certain sections of this chain may possess no biological 

 function. This question will be discussed later (see Section III, 1, e). 



Furthermore, it has not yet been proved that all 6000 nucleotides are 

 bonded to each other through covalent bonds (phosphoric acid diester 

 bonds between neighboring nucleosides). Sensitive areas in the polynucleo- 

 tide chain, for example, labile phosphotriester bonds, are not excluded. 

 However, if subunits of the RNA should exist, these can be biologically 

 active only when they are all bonded together in the specific arrangement 

 of an RNA molecule of molecular weight 2 X 10 6 . 



(3) Chemical Approaches to the Structure of RNA. It has not yet been 



