Specific Mechanisms of Protein Synthesis in the Developing Chick Embryo 125 



machinery, which set them apart from otlier cells of higher organisms. These 

 are: the capacity for replication, that is, rapid yet controlled growth; the 

 capacity for differentiation, that is, continuous yet controlled change and 

 evolution (1). Therefore, one might consider this the system of choice for 

 attempts at discovering how the information content of the hereditary material, 

 the genetic potentialities, are translated into progressive biochemical capabilities 

 and thus into physiological and morphological realities (2). The experiments 

 were done with chick embryos in ovo because of the ease of handling and the 

 essentially closed and self-contained nature of the experimental system. Further- 

 more, there is a relative paucity of reliable, modern information available 

 about their metabolism and that of embryos of higher vertebrates in general, 

 as contrasted to the large body of knowledge derived from experimental 

 embryology. 



Our eventual aim is to study the initiation, the mode, and the control of 

 synthesis of highly specific, respiratory enzymes as an indicator of controlled 

 biosynthetic events; however, our initial investigations deal with the more 

 modest one of a definition of parameters for embryonic protein synthesis (3). 

 For any protein formed de novo, as has been pointed out by Spiegelman (4) 

 essentially three different mechanisms may be envisaged: 



1. The rearrangement of pre-existing protein molecules; namely, the 

 urprotein hypothesis of Northrop (5), with suitable modifications. 



2. The accretion of amino acids on to pre-existing proteins or peptides. 



3. De novo synthesis from amino acids. 



In the special case of the formation of induced enzymes in rapidly dividing 

 bacterial cells and cell-free systems derived therefrom, the evidence is over- 

 whelmingly in favor of the third alternative (4, 6). The situation is not nearly 

 as straightforward in the vertebrate systems studied. On the one hand, for 

 example, Work and collaborators investigated the synthesis of milk proteins 

 (7), Velick, Simpson and co-workers the synthesis of several specific enzyme 

 proteins for muscle (8, 9), and Loftfield and Harris the synthesis of liver 

 ferritin (10). All this work was in vivo and by different experimental techniques, 

 but all these authors presented strong evidence for the last alternative and against 

 the first two. On the other hand Anfinsen and his co-workers, working with 

 hen's oviduct in vitro, have demonstrated that in short term incubations 

 incorporation of amino acids into freshly formed ovalbumin is non-uniform, 

 which is suggestive of the second alternative, but that after longer periods 

 there is a redistribution towards unifonnity (11). Similar results have also 

 been obtained for ribonuclease and insulin synthesis by pancreas sHces. 



In the case of the proteins of the chick embryo proper, Francis and Winnick 

 have presented data on the incorporation of labeled amino acids in free and 

 protein-bound form as possible precursors of cardiac muscle protein grown 

 in tissue culture (12). The amino acids of the proteins did not exchange with 

 large pools of the corresponding unlabeled acid in the medium, and from this 

 and from experiments with doubly-labeled proteins it was concluded that 

 proteins could be transferred from a nutrient embryo extract medium to 

 heart muscle protein without release of free amino acids. Tracer experiments 

 of this sort, as will be discussed later, do not, however, prove the direct transfer 



