MECHANISMS OF GENE ACTION 295 



In further studies of the ad^ mutants, in vitro eomplementation was 

 discovered. By freezing and grinding together the separately grown 

 myceha of a pair of complementing strains, some enzyme activity was 

 recovered in the homogenate. The occurrence of in vitro complementa- 

 tion raises more questions than it answers. The process probably in- 

 volves gene products rather than the genes themselves, since it occurs in 

 the structurally disorganized mycelial homogenate. However, one can 

 only speculate about this at present, for no evidence is yet available 

 concerning the mechanism of the process. 



A possible mechanism of complementation has been proposed by 

 Singer and Itano, who have been studying in vitro recombination be- 

 tween the polypeptide chains of hemoglobin. They found that the four 

 polypeptide chains could be dissociated asymetrically, the two a-chains 

 separating from the two /3-chains, as shown in Figure 10.12, and that 

 subsequently these nonidentical halves could be induced to reassociate. 



The reassociation process has been investigated by labeling the poly- 

 peptide chains in a number of different ways. Here we shall limit our- 

 selves to a single example, in which genetically altered hemoglobins 

 were employed. The chemist's terminology and the net charge differ- 

 ences of these hemoglobins are: 



HbA 02-^/32"^ Neutral 



HbS a2^02^ 2 



Hhl a J 02^ -4 



The superscript A refers to normal hemoglobin, and the other super- 

 scripts refer to the abnormal types; this notation scheme makes clear 

 in which chain the abnormality is located. 



Because of the charge differences between these three types of hemo- 

 globin, they can be separated from one another by electrophoresis. In 



FIGURE 10.12. Recombination of hemoglobin polypeptides (after Itano, 1960, Acad. 

 Sc/., New York, 88:642). 



A. The possible types of dissociation of hemoglobin subunits are three: sym- 

 metrical dissociation as in types la and lb, and asymmetrical dissociation as in type II. 

 Under the conditions of this experiment, only type-ll dissociation occurred, as demon- 

 strated below 



6. Electrophoretic diagrams showing the charge distribution of hemoglobin pro- 

 teins, (o). A mixture of HbA and Hbl which diflFer by four net charges, (b). The same 

 mixture after dissociation and reassociation, showing that only type-ll dissociation 

 occurred, (c). A mixture of HbA, HbS, and Hbl before dissociation, (d). The same 

 mixture after reassociation, showing an increase in HbA, a decrease in Hbl, and the 

 presence of a new Hb with a net charge of — 2. These changes result from random re- 

 combination of ct-chains with /3-chains following asymmetrical dissociation (type II). 



