The Molecular Basis of Mutation 



393 



H 2 N 



*N' 



NH, 



Proflavin 



figure 31—1. Two acridine dyes. 



H,C 



N 



H,C 



TM' 



Acridine Orange 



.CH 



N 

 I 

 CH 3 



infection, in the incorporation of this base 

 analog in T4 DNA, since thymine can be 

 synthesized by the bacterium and it — rather 

 than the analog — may be used preferentially 

 or exclusively in the synthesis of phage DNA. 

 Sulfanilamide, itself not mutagenic, inhibits 

 synthesis of folic acid, which in its reduced 

 form (tetrahydrofolic acid) is required for 

 enzymatic methyl transfer reactions. There- 

 fore, sulfanilamide is added to the culture 

 medium to assure that no thymine is synthe- 

 sized from uracil. The medium is supple- 

 mented with a variety of essential chemical 

 substances already containing methyl and 

 hydroxymethyl groups but not with the de- 

 oxyribotides of thymine or of 5-hydroxy- 

 methyl cytosine. (The deoxyribotide of 5- 

 hydroxymethyl cytosine is omitted to prevent 

 its possible conversion to an analog of thy- 

 mine which might be incorporated in prefer- 

 ence to the 5-bromo uracil.) In this way, 

 the bacterium can function properly as a 

 phage host. 



Under these conditions, 5-bromo uracil is 

 highly mutagenic in the rll region. A com- 

 parison of 5-bromo uracil-induced and spon- 

 taneously-occurring rll mutants reveals that 

 the induced mutants also occur in clusters 

 on the genetic map, although the hot spots 

 are in different positions. Moreover, con- 

 trary to the spontaneous mutants, very few 

 of those induced are of the gross (internu- 

 cleotide) type, and almost all are subse- 

 quently capable of reverse mutation to, or 

 near the r+ phenotype. 



Although the mutational spectra (p. 192) 

 for 5-bromo uracil, other chemical mutagens. 



and spontaneous mutants are all different 

 at the nucleotide level, the exact chemical 

 basis for the induced mutations cannot be 

 specified with any certainty, because any 

 given mutagen may be producing its effect 

 via any of several different metabolic path- 

 ways. Clearly, the molecular basis for muta- 

 genic action is best studied using the shortest 

 possible physical pathway between chemical 

 mutagen and gene. 4 Thus, it is preferable 

 to treat sperm rather than oocytes with a 

 chemical mutagen, and more desirable to 

 expose phage or transforming DNA to the 

 mutagen directly, rather than indirectly, via 

 its host. 



Mutation Involving Whole Nucleotides 



Since the genetic material is a linear array 

 of nucleotides, let us consider the possible 

 changes to whole nucleotides at the basis of 

 mutations. One or more nucleotides can be 

 added, deleted, substituted for, inverted, or 

 transposed to a new position with or without 

 inversion. All these nucleotide rearrange- 

 ments ought to be possible for single- 

 stranded nucleic acid, except inversion, 

 which requires double-stranded nucleic acid 

 to maintain strand polarity. Whole-nucleo- 

 tide changes can be produced by breaking 

 the polynucleotide backbone at two or more 

 places, followed by rearrangement of the 

 fragments. Breakage of the backbone (and 

 loss of DNA's ability to act as primer-tem- 

 plate) occurs especially often after expo- 

 sure to an ionizing physical mutagen, and 

 leads to deletions and other rearrangements 



4 As noted by I. H. Herskowitz (1955). 



