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CHAPTER 49 



ship between cistrons and polypeptides is 

 also expected to be extremely useful for de- 

 termining the details of the DNA and RNA 

 codes, and the nature of mutation. We 

 have discussed several genetic systems which 

 seem of special interest in these respects. 

 One involves the genetic determination of 

 hemoglobin. In this case, however, while 

 the amino acid sequence of some of the pro- 

 tein is known, it is a difficult undertaking to 

 determine the corresponding nucleotide se- 

 quence because of the large amount of nu- 

 cleotide material in the genome. Several 

 other systems should be mentioned. All 

 involve viruses, which have a relatively small 

 number of nucleotides per genome. 



The amino acid sequence in the protein 

 building block of tobacco mosaic virus is 

 now known (see Figure 44-4). We need 

 now to determine the sequence of ribotides 

 in TMV. This is a formidable undertaking 

 technically, whose success is hoped for, but 

 is not assured. Progress along these lines is 

 evidenced ^ by the finding that the termi- 

 nal 5' linked nucleotide in isolated TMV- 

 RNA is adenosine, unphosphorylated at the 

 2' and 3' positions. Another line of attack 

 attempts to correlate the details of the pro- 

 tein coat of phage with phage nucleotide 

 sequence. 



Still another line of investigation ^ involves 

 the rll cistron in phage, whose fine structure 

 is analyzable down to the nucleotide level, 

 but whose phenotypic effect is not under- 

 stood in detail chemically. Nevertheless, it 

 is possible to determine the nucleotide basis 

 for certain point mutants in the rll region. 

 Suppose, in r+, that the DNA strand "mak- 

 ing" messenger RNA has a G in a certain 

 triplet, and that this is replaced by A in a 

 particular r point mutant. This mutant 



2 From work of T. Sugiyama and H. Fraenkel- 

 Conrat, in 1961. 



3 Pursued by S. Benzer with S. P. Champe and other 

 coworkers, and by others. (See reference in legend 

 to Figure 43-5.) 



phage may not lyse the K12 strain of E. coli 

 because its messenger RNA is abnormal and 

 contains U instead of C, and wrongly made 

 /•+ product results. It has been found that 

 5-fluoro uracil, FU, is not mutagenic, but 

 can substitute for the U in RNA when added 

 to the diet of K12. When FU substitutes for 

 U in messenger RNA, the FU may some- 

 times be mistaken for C by an sRNA mole- 

 cule. So, in the case of the mutant under 

 discussion, the sRNA paired with abnormal 

 messenger RNA may be wrong, but it may 

 be the one which carries the amino acid 

 brought to this position in normal, r+, mes- 

 senger RNA. As a result, the normal amino 

 acid would be incorporated, r+ product 

 would be formed, and the host cell would lyse. 

 So, those r mutants, which can lyse only 

 when FU is added, most probably have G 

 on the /•+ DNA strand which "makes" mes- 

 senger RNA, and C on the complementary 

 DNA strand. Those mutants which do not 

 lyse in the presence of FU may have T, A, or 

 C at this locus in the DNA strand "making" 

 messenger RNA. Using various chemical 

 mutagens as well as FU, it is often possible 

 to determine when T, or A, or C is pres- 

 ent on the /•+ messenger-producing DNA 

 strand. 



It is sometimes found that a single phage 

 mutant may simultaneously suppress the ef- 

 fects of point mutants at a number of other 

 nucleotide sites. Suppose, in some of these 

 cases, that all the latter point mutants have 

 the same triplet modified by the same base 

 substitution, and the result is that the same 

 abnormal amino acid is incorporated into the 

 different cistronic products. One way to 

 suppress the effects of all of these mutants 

 would be for a mutation to occur in the DNA 

 which specifies the enzyme which determines 

 which amino acid is transported by the 

 specific sRNA which pairs with the abnormal 

 triplet in the messenger RNA. In this case, 

 an abnormal amino acid may be transported 

 to the abnormal messenger RNA, and this 



