The Origin of Specific Proteins 111 



several nuclear fractions produced these effects, we are not sure of the 

 identity of the effective suhstances, although they appear to he non- 

 dialyzahle proteins that are inactivated hy prolonged heating. The 

 most potent fraction is still active when diluted, so that its protein 

 content is as little as 0.02 millimicrograms of protein per 0.1 micro- 

 liter — the volume of the injected solution. Further dilution rendered 

 this solution ineffective. During cell division the chromosomes of in- 

 jected eggs should have heen freely accessihle to any injected sub- 

 stances. The very small quantity of injected material suggests a highly 

 specific effect. Moreover, when nuclei from arrested hlastulas were 

 serially transplanted for seven generations (Fig. 7 I to enucleated eggs, 

 the embryos that resulted invariably stopped developing before the 

 onset of gastrulation ( Markert and Ursprung, 1962 ) . Since the effect 

 was very characteristic and persistent through many nuclear divisions, 

 it seems reasonable to place the primary responsibility on the chromo- 

 somes. The behavior of these nuclei 'closely resembles that of the 

 transplanted nuclei which multiplied in foreign cytoplasm (Moore, 

 1960 I , and indeed the chromosomes of the injected eggs were initially 

 multiplying in "foreign" cytoplasm — a cytoplasm changed by virtue 

 of the injected substances from the adult liver nuclei. 



Isozymes 



The discussion so far has hopefully revealed the importance of the 

 cytoplasm in activating gene function, an event that is manifested in 

 the synthesis of a specific protein. Is this then an inclusive description 

 of the mechanisms underlying the synthesis of specific proteins? 

 Probably not. Embryologists have often loosely spoken of molecular 

 differentiation when they really meant the differentiation of cells as 

 evidenced by the appearance of new proteins. However, we have 

 recently become aware of a phenomenon that may be a valid example 

 of a true molecular differentiation. Many investigators have shown 

 that single enzymes commonly exist in multiple molecular forms or 

 isozymes within the tissues or cells of a single organism (Markert and 

 M0ller, 1959 ) . The isozymes of a single enzyme have very similar 

 catalytic properties but can nevertheless be distinguished from one 

 another by their somewhat different physical properties. Usually 

 chromatographic or electrophoretic techniques serve to separate iso- 

 zymes from one another. These isozymes are not artifacts of analysis 

 but exhibit characteristic patterns of distribution in each, tissue (Fig. 

 8). Moreover, the tissue patterns are species specific (Fig. 9). The 

 characteristically different isozyme patterns of adult tissues must have 



