Chapter *44 



VIRUSES: BACTERIAL, ANIMAL, 

 AND PLANT 



JLmc 



"n discussing the genetics of the 

 rll region of the T4 phage genetic 

 lap, it was mentioned (p. 394) 

 that the more than 1500 spontaneously oc- 

 curring mutants tested could be explained as 

 involving changes in one or more of about 

 300 different sites in the rll region. This 

 means that some sites of mutation must have 

 been involved more than once. In fact, the 

 number of times different sites were involved 

 in mutation is quite variable. In terms of 

 DNA this must mean that certain nucleo- 

 tides, singly or in groups, are much more 

 Hkely to undergo spontaneous mutation than 

 others, there being, so to speak, mutational 

 '''hot spots" within the rll region. 



Since recombination studies permit the 

 analysis of the rll region at the level of the 

 nucleotide, the DNA of T4 may serve as 

 material for studies on the nature of mutation 

 which may lead to a clearer definition of 

 mutation in chemical terms. It should be 

 noted at this point that even-number T phages 

 (T2, T4, T6) have 5-hydroxymethyl cytosine 

 (Figure 33-2, and p. 296) instead of cytosine 

 in their DNA. In all other respects, the 

 DNA is typical. It was already noted, on 

 page 325, that 5-bromo uracil (Figure 35-5) 

 can substitute for thymine, and only thymine, 

 in the synthesis of DNA in vitro. What 

 would be the mutational consequence of in- 

 corporating 5-bromo uracil into T4 DNA? ^ 



1 The discussion following is based largely upon the 

 work of S. Benzer and E. Freese (1958) and subse- 

 quent work by E. Freese and coworkers. 

 400 



Addition of 5-bromo uracil to the normal cul- 

 ture medium in which T4-infected cells are 

 growing would not necessarily result in the in- 

 corporation of this base analog in T4 DNA, 

 since thymine could be synthesized by the 

 bacterium and it, rather than the analog, 

 might be utilized preferentially or exclusively 

 in the synthesis of phage DNA. In order to 

 assure that no thymine is synthesized or 

 available as raw material for DNA synthesis, 

 sulfanilamide is added to the culture medium. 



This drug, which by itself does not appreci- 

 ably increase the mutation rate, inhibits 

 nearly all syntheses leading to the addition 

 of methyl or hydroxymethyl groups to com- 

 pounds. Accordingly, the medium is sup- 

 plemented with a variety of essential chemical 

 substances already containing methyl and 

 hydroxymethyl groups but not with the 

 deoxyribotides of thymine or of 5-hydroxy- 

 methyl cytosine. (The latter deoxyribotide 

 is omitted to prevent its possible conversion 

 to an analog of thymine which might be 

 incorporated in preference to the 5-bromo 

 uracil.) In this way, the bacterium can prop- 

 erly function as a phage host, but cannot, for 

 example, produce thymine (5-methyl uracil) 

 by methylating the uracil which is present in 

 abundance in the cytoplasm and its RNA, 

 Under these conditions, then, 5-bromo uracil 

 will be used as a substitute for thymine in 

 DNA synthesis. It should be noted, however, 

 that the base analog may also be falsely 

 incorporated in place of 5-hydroxymethyl 

 cytosine, or act in other ways, in producing a 

 mutagenic effect. 



Under these experimental conditions, it is 

 found that 5-bromo uracil is highly muta- 

 genic in the rll region. A comparison of 

 5-bromo uracil-induced and spontaneously 

 occurring rll mutants reveals that the induced 

 mutants also occur in clusters on the genetic 

 map, but that the hot spots are in different 

 positions. Moreover, contrary to the spon- 

 taneous mutants, very few of those induced 

 are of gross (internucleotide) type, and almost 



