392 



CHAPTER 31 



The spontaneous mutation rate is reduced 



bj the presence of purine ribosides | adeno- 

 sine or guanosine) but not pyrimidine ribo- 

 sides (uridine or cytidine) in the medium. 



With adenosine in the medium, the sponta- 

 neous rate is reduced to about one third its 

 original value. Moreover, the spontaneous 

 rate is lower under anaerobic than aerobic 

 conditions, as would be expected from the 

 metabolic production or lack of utilization 

 of adenosine. Finally, it should be noted 

 that all the purine mutagens increase the 

 mutation rate to T5 resistance more than to 

 T6 resistance, although the reverse is true 

 for ultraviolet and gamma radiation. 



From these results it seems reasonable to 

 distinguish two kinds of mutagens — a purine 

 type and a radiation type — which produce 

 two different kinds of mutations. We can 

 postulate that under the experimental condi- 

 tions described, about two thirds of the spon- 

 taneous mutation rate is produced by the 

 action of some purine type of substance pro- 

 duced spontaneously during the normal me- 

 tabolism of the cell. It may seem surprising 

 that only purines, their analogs, and purine 

 ribosides affect the mutation rate. This find- 

 ing, however, may be related to the par- 

 ticular mutants studied — namely, those re- 

 sistant to phages T5 or T6. Such muta- 

 tions may depend more upon changes in 

 purines than pyrimidines; other mutations 

 may prove to be relatively pyrimidine-sensi- 

 tive and purine-insensitive. 



Even though it is not clear how the pu- 

 rines and their ribosides accomplish their 

 mutagenic and antimutagenic effects, two 

 general conclusions are warranted: 



1 . A considerable portion of the sponta- 

 neous mutation rate is the normal con- 

 sequence of the cell's biochemical 

 activity in producing mutagens and 

 antimutagens. 



2. A connection exists between mutation 

 rate and nucleic acid metabolism. 



I hough the mutation rate is directly con- 

 nected only with purines and their ribosides, 

 supported h\ the fact that thymine is muta- 

 genic when withheld from bacteria requir- 

 ing it. it is reasonable to suppose that an 

 indirect connection also exists with DNA 

 and its precursors. 



Mutational Spectra 



In discussing the genetics of the rll region 

 of the 4>T4 genetic map, it was mentioned 

 (p. 345) that the more than 1500 sponta- 

 neously-occurring mutants tested involved 

 changes in one or more of about 300 diller- 

 ent sites in the rll region. This statement. 

 of course, implies that some mutation sites 

 must have been involved more than once. 

 In fact, the number of times different sites 

 are involved in mutation varies considerably. 

 In terms of DNA, this variability must mean 

 that certain nucleotides, singly or in groups, 

 are much more likely to undergo sponta- 

 neous mutation than others, so that muta- 

 tional "hot spots" must occur. 



Since recombination studies permit the 

 analysis of the rll region at the level of the 

 nucleotide, the DNA of T4 can serve as ma- 

 terial for studies leading to a clearer defini- 

 tion of mutation on the molecular level. 

 Note that even-number T phages (T2, T4, 

 T6) have 5-hydroxymethyl cytosine (Figure 

 19-3 on p. 255) or a derivative of it, in- 

 stead of cytosine in their DNA; in all other 

 respects this DNA is typical. It has already 

 been noted (p. 283) that 5-bromo uracil 

 (Figure 21-4) can substitute for thymine — 

 and only thymine — in the synthesis of DNA 

 in vitro. What will be the mutational conse- 

 quences of incorporating 5-bromo uracil into 

 T4 DNA.' 



Addition of 5-bromo uracil to the normal 

 culture medium of E. coli before infection 

 with T4, does not necessarily result, alter 



3 The discussion Following is based largely upon 

 the work of S. Benzer and E. Freese (1958), and 

 subsequent work by E. Freese and co-workers. 



